Makrofagų aktyvacijos molekulinių mechanizmų tyrimai.

Macrophages are innate immune cells responsible for defence and homeostasis. One of the innate immunity defence mechanisms is inflammasome activation, which is also related to many inflammatory diseases. NLRP3 inflammasome can be triggered by a variety of factors. However, inflammasome activation by protein oligomers is less investigated. Our goal was to investigate macrophage activation by oligomeric proteins of various structures. We treated macrophages by these proteins: amyloid beta (Aβ) small oligomers and protofibrils; filamentous N proteins of paramyxoviruses; spherical virus-like particles (VLP) of polyomaviruses. We measured inflammatory cytokine secretion and NLRP3 inflammasome activation by assessing IL-1β secretion, caspase-1 activation and formation of ASC specks. We found that both Aβ structures activated inflammasome. This shows the probability that immune response arises before accumulation of Aβ fibrils during Alzheimer’s disease. Therefore, primary Aβ structures could be a source of neuroinflammation. Investigation of viral proteins showed that only VLP induced inflammatory response and activated inflammasome. VLP induced lysosomal damage and K+ ion efflux mediating inflammasome activation. Our investigation demonstrates that the structure of oligomeric proteins is one of the factors defining inflammatory response. Moreover, our results highlight NLRP3 inflammasome role in macrophage response to various molecular structures.Macrophages are innate immune cells responsible for defence and homeostasis. One of the innate immunity defence mechanisms is inflammasome activation, which is also related to many inflammatory diseases. NLRP3 inflammasome can be triggered by a variety of factors. However, inflammasome activation by protein oligomers is less investigated. Our goal was to investigate macrophage activation by oligomeric proteins of various structures. We treated macrophages by these proteins: amyloid beta (Aβ) small oligomers and protofibrils; filamentous N proteins of paramyxoviruses; spherical virus-like particles (VLP) of polyomaviruses. We measured inflammatory cytokine secretion and NLRP3 inflammasome activation by assessing IL-1β secretion, caspase-1 activation and formation of ASC specks. We found that both Aβ structures activated inflammasome. This shows the probability that immune response arises before accumulation of Aβ fibrils during Alzheimer’s disease. Therefore, primary Aβ structures could be a source of neuroinflammation. Investigation of viral proteins showed that only VLP induced inflammatory response and activated inflammasome. VLP induced lysosomal damage and K+ ion efflux mediating inflammasome activation. Our investigation demonstrates that the structure of oligomeric proteins is one of the factors defining inflammatory response. Moreover, our results highlight NLRP3 inflammasome role in macrophage response to various molecular structures

Immunogenic Properties and Antigenic Similarity of Virus-like Particles Derived from Human Polyomaviruses

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system

Investigation of the impact of different oligomeric proteins and their immune complexes on the phenotype of macrophages

Investigation of the Influence of Various Origin and Structure Oligomeric Proteins and Their Immune Complexes on Macrophage Phenotype Macrophages are a part of organism defence system. They remove pathogenic microorganisms and cellular debris and may give an activation signal to other cells of the immune system. They also can detect antigens opsonized with antibodies and internalize them. Therefore, macrophages might have an impact on the efficiency of antigen elimination in certain diseases. An example is microglia-mediated removal of β-amyloid (Aβ) oligomers in Alzheimer's disease (AD). However detailed mechanism of macrophage activation by the immune complexes (IC) is unknown as certain side effects of anti-Aβ therapy have been shown in treatment of AD. The aim of the current study was to investigate the influence of Aβ oligomers and viral oligomers (measles virus, WU polyomavirus and metapneumovirus) and their IC on macrophage phenotype. The research model was primary spleen cell culture from BALB/c mice. The macrophages were generated from splenic monocytes incubated with macrophage colony stimulating factor. The levels of different cellular markers indicative of either inflammatory macrophage M1 phenotype or anti-inflammatory macrophage M2 phenotype were investigated by flow cytometry and ELISA. Induction of both M1 and M2 phenotype after macrophage treatment with Aβ oligomers and their IC was demonstrated. The IC induced higher expression of M2-related marker IL-10 compared to Aβ oligomers alone. Higher levels of IL-10 may indicate a negative impact of IC on the phagocytic degradation of Aβ oligomers. Macrophage treatment with oligomeric viral antigens and their IC induced the inflammatory phenotype of macrophages. However, the activation signal was lower compared to Aβ oligomers. In conclusion, the activation of macrophages with oligomeric proteins and their IC can induce different signals and lead either to the inflammatory or anti-inflammatory phenotype depending on the origin and structure of the oligomeric proteins

Activation of Macrophages by Oligomeric Proteins of Different Size and Origin

Activation of macrophages is one of the key processes in generating the immune response against pathogens or misfolded/aggregated otherwise unharmful host’s proteins. Antigens and their immune complexes (IC) may shape macrophage phenotype in various directions. Data on the impact of protein structure during inflammation are evident; however, some separate steps of this process involving changes in macrophage phenotype are not fully understood. Our aim was to investigate the phenotype of macrophages after activation with different oligomeric proteins and their IC. We have used amyloid beta (Aβ1–42) that plays a role in neurodegenerative inflammation as a model of host-associated protein and three oligomeric viral antigens as pathogen-associated proteins. Murine cell lines J774, BV-2, and macrophage primary cell culture were treated with oligomeric proteins and their IC. After 48 h, expression of surface markers F4/80, CD68, CD86, and CD206 and secreted cytokines IL-10, IL-12, IL-23, and TNF-α was analysed. Aβ1–42 oligomers stimulated expression of both inflammatory and anti-inflammatory molecules; however, fibrils induced less intense expression of markers investigated as compared to small and large oligomers. Two out of three viral oligomeric proteins induced the inflammatory response of macrophages. Data suggest that macrophage activation pattern depends on the origin, size, and structure of oligomeric proteins

OmpA Protein-Deficient Acinetobacter baumannii Outer Membrane Vesicles Trigger Reduced Inflammatory Response

Multidrug resistant Acinetobacter baumannii shows a growing number of nosocomial infections worldwide during the last decade. The outer membrane vesicles (OMVs) produced by this bacterium draw increasing attention as a possible treatment target. OMVs have been implicated in the reduction of antibiotic level in the surrounding environment, transfer of virulence factors into the host cells, and induction of inflammatory response. Although the evidence on the involvement of OMVs in A. baumannii pathogenesis is currently growing, their role during inflammation is insufficiently explored. It is likely that bacteria, by secreting OMVs, can expand the area of their exposure and prepare surrounding matrix for infection. Here, we investigated the impact of A. baumannii OMVs on activation of macrophages in vitro. We show that OmpA protein present in A. baumannii OMVs substantially contributes to the proinflammatory response in J774 murine macrophages and to the cell death in both lung epithelium cells and macrophages. The loss of OmpA protein in OMVs, obtained from A. baumannii ∆ompA mutant, resulted in the altered expression of genes coding for IL-6, NLRP3 and IL-1β proinflammatory molecules in macrophages in vitro. These results imply that OmpA protein in bacterial OMVs could trigger a more intense proinflammatory response

Immunogenic properties and antigenic similarity of virus-like particles derived from human polyomaviruses /

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system

Structural properties of immune complexes formed by viral antigens and specific antibodies shape the inflammatory response of macrophages

Abstract Data on the course of viral infections revealed severe inflammation as a consequence of antiviral immune response. Despite extensive research, there are insufficient data on the role of innate immune cells in promoting inflammation mediated by immune complexes (IC) of viral antigens and their specific antibodies. Recently, we demonstrated that antigens of human polyomaviruses (PyVs) induce an inflammatory response in macrophages. Here, we investigated macrophage activation by IC. We used primary murine macrophages as a cell model, virus-like particles (VLPs) of PyV capsid protein as antigens, and a collection of murine monoclonal antibodies (mAbs) of IgG1, IgG2a, IgG2b subclasses. The inflammatory response was investigated by analysing inflammatory chemokines and activation of NLRP3 inflammasome. We observed a diverse pattern of chemokine secretion in macrophages treated with different IC compared to VLPs alone. To link IC properties with cell activation status, we characterised the IC by advanced optical and acoustic techniques. Ellipsometry provided precise real-time kinetics of mAb-antigen interactions, while quartz crystal microbalance measurements showed changes in conformation and viscoelastic properties during IC formation. These results revealed differences in mAb-antigen interaction and mAb binding parameters of the investigated IC. We found that IC-mediated cell activation depends more on IC characteristics, including mAb affinity, than on mAb affinity for the activating Fc receptor. IC formed by the highest affinity mAb showed a significant enhancement of inflammasome activation. This may explain the hyperinflammation related to viral infection and vaccination. Our findings demonstrate that IC promote the viral antigen-induced inflammatory response depending on antibody properties

Activation of NLRP3 inflammasome by virus-like particles of human polyomaviruses in macrophages

Viral antigens can activate phagocytes, inducing inflammation, but the mechanisms are barely explored. The aim of this study is to investigate how viral oligomeric proteins of different structures induce inflammatory response in macrophages. Human THP-1 cell line was used to prepare macrophages that were treated with filamentous nucleocapsid-like particles (NLPs) of paramyxoviruses and spherical virus-like particles (VLPs) of human polyomaviruses. The effects of viral proteins on cell viability, pro-inflammatory cytokines' production, and NLRP3 inflammasome activation were investigated. Filamentous NLPs did not induce inflammation while spherical VLPs mediated inflammatory response followed by NLRP3 inflammasome activation. Inhibitors of cathepsins and K+ efflux decreased IL-1β release and cell death, indicating a complex inflammasome activation process. A similar activation pattern was observed in primary human macrophages. Single-cell RNAseq analysis of THP-1 cells revealed several cell activation states different in inflammation-related genes. This study provides new insights into the interaction of viral proteins with immune cells and suggests that structural properties of oligomeric proteins may define cell activation pathways

Soluble Aβ oligomers and protofibrils induce NLRP3 inflammasome activation in microglia

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder causing memory loss, language problems and behavioural disturbances. AD is associated with the accumulation of fibrillar amyloid-β (Aβ) and the formation of neurofibrillary tau tangles. Fibrillar Aβ itself represents a danger-associated molecular pattern, which is recognized by specific microglial receptors. One of the key players is formation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome, whose activation has been demonstrated in AD patient brains and transgenic animal models of AD. Here, we investigated whether Aβ oligomers or protofibrils that represent lower molecular aggregates prior to Aβ deposition are able to activate the NLRP3 inflammasome and subsequent interleukin-1 beta (IL-1β) release by microglia. In our study, we used Aβ preparations of different sizes: small oligomers and protofibrils of which the structure was confirmed by atomic force microscopy. Primary microglial cells from C57BL/6 mice were treated with the respective Aβ preparations and NLRP3 inflammasome activation, represented by caspase-1 cleavage, IL-1β production, and apoptosis-associated speck-like protein containing a CARD speck formation was analysed. Both protofibrils and low molecular weight Aβ aggregates induced a significant increase in IL-1β release. Inflammasome activation was confirmed by apoptosis-associated speck-like protein containing a CARD speck formation and detection of active caspase-1. The NLRP3 inflammasome inhibitor MCC950 completely inhibited the Aβ-induced immune response. Our results show that the NLRP3 inflammasome is activated not only by fibrillar Aβ aggregates as reported before, but also by lower molecular weight Aβ oligomers and protofibrils, highlighting the possibility that microglial activation by these Aβ species may initiate innate immune responses in the central nervous system prior to the onset of Aβ deposition