Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

BACKGROUND: The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcgammaR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcgammaR and the Fc domain of IgG depend on the IgG glycosylation state. METHODOLOGY/PRINCIPAL FINDINGS: Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcgammaR and to an erythroleukemic cell line, K562, expressing FcgammaRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcgammaRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. CONCLUSIONS/SIGNIFICANCE: We provide novel information about bacterial enzymatic modulation of the IgG/FcgammaR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes

Oral administration of the selective GPR120/FFA4 agonist compound A is not effective in alleviating tissue inflammation in mouse models of prototypical autoimmune diseases

ω3-polyunsaturated free fatty acids (ω3-PUFAs), particularly docosahexaenoic (DHA) and eicosapentaenoic acid (EPA), are thought to exert health promoting effects in metabolic and in inflammatory diseases. The molecular mechanisms of these beneficial effects are only partially understood. DHA and EPA activate Free Fatty Acid receptor 4 (GPR120/FFA4). Recently, the first orally available, synthetic ligand of FFA4, 3-[2-chloro-5-(trifluoromethoxy)phenyl]-3-azaspiro[5.5]undecane-9-acetic acid ("compound A"; cpd A) has been developed. Cpd A exhibits distinctly higher potency, efficiency, and selectivity at FFA4 than ω3-PUFAs and ameliorates insulin resistance and adipose tissue inflammation in the mouse. With GPR120/FFA4 activation believed to also attenuate tissue inflammation in autoimmune diseases, cpd A may also have a beneficial effect in these diseases. We have therefore addressed the therapeutic potential of cpd A in mouse models of three prototypical autoimmune diseases, specifically psoriasis, rheumatoid arthritis, and bullous pemphigoid. The effect of cpd A on the course of Aldara™-induced psoriasis-like dermatitis, K/BxN serum transfer arthritis, and antibody transfer pemphigoid disease-like dermatitis was scrutinized. Cpd A did not alter the course of Aldara-induced psoriasis-like dermatitis, K/BxN serum transfer arthritis, or antibody transfer pemphigoid disease-like dermatitis. Our results suggest that therapeutic regimens solely relying on FFA4 activation do not bear the potential to treat inflammatory diseases. With cpd A distinctly more potent in activating GPR120/FFA4 than ω3-PUFAs, this also suggests that GPR120/FFA4 activation by ω3-PUFAs does not significantly contribute to the health-promoting effects of ω3-PUFAs in autoimmune diseases

Polymeric human Fc-fusion proteins with modified effector functions

The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored

The state of the art: immune-mediated mechanisms of monoclonal antibodies in cancer therapy

A number of antibody products have now become accepted as effective anti-cancer therapies. Despite being mainly designed to act by inhibiting functional tumour antigens, there is increasing evidence that Fc-mediated engagement of the immune system is an important contributor to the efficacy of several of these therapies. The optimisation of this engagement offers the potential not only to augment efficacy against existing targets, but also to exploit non-functional tumour antigens. Antibodies that achieve efficacy wholly or predominantly through Fc-mediated mechanisms, represent rich opportunities for future therapeutics in oncology. This mini review summarises some of the key challenges, which need to be addressed to select the most effective molecules. These include the identification of optimal antibody characteristics and improvement of the drug discovery process, in particular, the relevance and predictive power of existing in vitro and in vivo screening methods. Advances in our understanding of tumour immunobiology and successful application of technologies designed to enhance immune system engagement will further aid this process

Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-κB promoter activation

<p>Abstract</p> <p>Background</p> <p>Influenza virus infection activates NF-κB and is a general prerequisite for a productive influenza virus infection. On the other hand, non-structural protein 1 (NS1) suppresses this viral activated NF-κB, presumably to prevent expression of NF-κB mediated anti-viral response. NS1 proteins of influenza A viruses are divided into two groups, known as allele A and allele B. The possible functional relevance of this NS1 division to viral pathogenicity is lacking.</p> <p>Findings</p> <p>The ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by <it>in situ </it>PLA.</p> <p>Conclusions</p> <p>Here, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus's pathogenic potential.</p

Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-gamma), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO-), or using the ONOO- degradation catalyst FeTMPyP cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 mu M), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.Foundation for Science and Technology, (FCT, Portugal); COMPETE; FEDER [PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014, PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012]; FCT, Portugal [SERH/BPD/78901/2011, SERH/BD/38127/2007, SFRH/BD/77903/2011, SFRH/BD/79308/2011]info:eu-repo/semantics/publishedVersio