Cross-talk between pathogen recognizing Toll-like receptors and immunoglobulin Fc receptors in immunity

The individual role of pathogen-binding Toll-like receptors (TLRs) and antibody-binding Fc receptors (FcRs) during pathogenic infections has been studied extensively. However, combined activation of these different receptor classes has received little attention, even though they are triggered simultaneously when immune cells bind antibody-opsonized pathogens. In the last few years, it has become evident that joined activation of TLRs and FcRs substantially tailors inflammatory immune responses, which is an efficient and controlled mechanism of the host to act upon invading pathogens. In this review, we discuss the mechanisms of cross-talk between different TLRs and FcRs and the resulting inflammatory immune responses. Furthermore, we propose how chronic activation via this cross-talk might be detrimental in inflammatory (auto) immune diseases. We conclude with the potential exploitation of the interplay between TLRs and FcRs for monoclonal antibody therapy to target tumors. Future interests in this field of research include establishing a more detailed and mechanistic understanding of the mode of action of TLR and FcR cross-talk and exploration of its physiological importance in health and disease. This may furthermore open up novel therapeutic options for intervention in inflammatory diseases or cance

IgA enhances NETosis and release of neutrophil extracellular traps by polymorphonuclear cells via Fcα receptor I

Polymorphonuclear cells (neutrophils) are the first cells that arrive at sites of infections. According to the current dogma, they are involved in eliminating bacteria, after which they die through apoptosis. We now demonstrate that enhanced IgA-induced phagocytosis of bacteria or beads by neutrophils led to increased cell death. Nuclear changes and positivity for the general cell death marker 7-aminoactinomycin D were observed, but the absence of annexin V membrane staining supported that neutrophils did not die via apoptosis, in contrast to neutrophils that had not phagocytosed bacteria. Moreover, increased release of neutrophil extracellular traps (NETs) was observed, which was most likely due to augmented production of reactive oxygen species after uptake of IgA-opsonized particles. Blocking the IgA Fc receptor FcαRI abrogated phagocytosis and NET formation. Thus, FcαRI triggering on neutrophils resulted in a rapid form of cell death that is referred to as NETosis, as it is accompanied by the release of NETs. As such, IgA may play a prominent role in mucosal inflammatory responses, where it is the most prominent Ab, because it enhanced both phagocytosis of bacteria and formation of NETs, which are effective mechanisms that neutrophils employ to eliminate pathogen

Protein 4.1G binds to a unique motif within the Fc-gamma RI cytoplasmic tail

The C-terminal domain of protein 4.1G was identified to interact with the cytosolic tail of the high affinity IgG receptor, Fc gamma RI, in yeast two-hybrid screens. Proteins of the 4.1 family have previously been found to mediate receptor/cytoskeleton interactions. In the study presented here, we show an alternatively spliced 4.1G product to be associated with increased Fc-gamma RI binding in yeast two-hybrid assays, and to be selectively enriched in most immune cells at the transcript level. In addition, a detailed analysis of the 4.1G 'docking site' within Fc gamma RI is provided by examining Fc gamma RI-CY-truncated and alanine-substituted mutants. These pointed to an Fc gamma RI membrane-proximal core motif of HxxBxxxBB (H represents hydrophobic residues, B basic residues and x represents any residue), followed by hydrophobic and (potentially) negatively charged residues to be central for interaction with protein 4.1G. (C) 2007 Elsevier Ltd. All rights reserved

Inside-out regulation of Fc alpha RI (CD89) depends on PP2A

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, Fc alpha RI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcaRI tail is important for F alpha aRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in Fc alpha RI activation. PP2A hinds to the intracellular tail of FcaRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, Fc alpha RI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcaRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis or Neisseria meningitidis. The present findings demonstrate MA involvement as a molecular mechanism for FcaRI ligand binding regulation, a key step in initiating an immune respons

FcαRI Dynamics Are Regulated by GSK-3 and PKCζ During Cytokine Mediated Inside-Out Signaling

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics

Antibody-opsonized bacteria evoke an inflammatory dendritic cell phenotype and polyfunctional th cells by cross-talk between TLRs and FcRs

During secondary immune responses, Ab-opsonized bacteria are efficiently taken up via FcRs by dendritic cells. We now demonstrate that this process induces cross-talk between FcRs and TLRs, which results in synergistic release of several inflammatory cytokines, as well as altered lipid metabolite profiles. This altered inflammatory profile redirects Th1 polarization toward Th17 cell responses. Interestingly, GM-CSF-producing Th cells were synergistically evoked as well, which suggests the onset of poly-functional Th17 cells. Synergistic cytokine release was dependent on activation via MyD88 and ITAM signaling pathways through TLRs and FcRs, respectively. Cytokine regulation occurred via transcription-dependent mechanisms for TNF-α and IL-23 and posttran scriptional mechanisms for caspase-1-dependent release of IL-1β. Furthermore, cross-talk between TLRs and FcRs was not restricted to dendritic cells. In conclusion, our results support that bacteria alone initiate fundamentally different immune responses compared with Ab-opsonized bacteria through the combined action of two classes of receptors and, ultimately, may refine new therapies for inflammatory diseases. Copyright © 2015 by The American Association of Immunologists, Inc

Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration

The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides’ half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases

Immunoglobulin A: Fc(alpha)RI interactions induce neutrophil migration through release of leukotriene B4

BACKGROUND & AIMS: Exacerbations of ulcerative colitis (UC) are dominated by massive neutrophil influx in the lamina propria with concomitant mucosal ulceration. The prevalent antibody in this area is immunoglobulin A (IgA). Interestingly, the IgA Fc receptor (Fc(alpha)RI) potently activates neutrophils. As such, we investigated whether IgA-Fc(alpha)RI interaction contributes to tissue damage in UC. METHODS: Response of neutrophils to bovine serum albumin-, IgG-, or IgA-coated beads and Escherichia coli was investigated with 3-dimensional culture systems, real-time video microscopy, and (fluorescence) microscopy. In vivo studies were performed using human Fc(alpha)RI transgenic mice or nontransgenic littermates. Microscopic slides of UC patients were stained for IgA, Fc(alpha)RI, and neutrophils. RESULTS: In vitro and in vivo cross-linking of Fc(alpha)RI on neutrophils by serum IgA or uptake of IgA-coated E coli led to neutrophil migration. The responsible chemotactic factor was identified as leukotriene B4. Moreover, dimeric IgA (dIgA), which is produced in the lamina propria, but neither secretory IgA nor IgG, was equally capable of inducing neutrophil recruitment. We furthermore showed that Fc(alpha)RI(+)-neutrophils in the colon of UC patients had phagocytosed IgA-antigen complexes. CONCLUSIONS: Neutrophils are the first cells that arrive at inflammatory sites once pathogens have crossed the epithelial barrier. Fc(alpha)RI-dIgA interactions therefore may constitute an essential activation step to recruit more neutrophils, hereby eradicating impending infections. However, excessive IgA-antigen complexes can sustain a perpetuating inflammatory loop in UC, hereby seriously aggravating morbidity. Novel therapeutic strategies that block dIgA-Fc(alpha)RI interactions, and therefore diminish neutrophil migration and activation, may dampen the uncontrolled inflammatory processes in these patient

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cell