Inhibition of Complement Pathways by Llama Nanobodies
Excessive activation of the complement lectin pathway (LP) and classical pathway (CP) is associated with multiple autoimmune and inflammatory diseases. In this Top Read, Zarantonello et al. (p. 1678) described a potent llama-derived C4b inhibiting nanobody (hC4Nb8). hC4Nb8 bound human C4b with picomolar affinity and potently inhibited complement C3 deposition through the CP and LP in both human and mouse serum. Analysis of the hC4Nb8:C4b complex crystal structure showed that hC4Nb8 binds to a neoepitope in C4b formed by residues in the αʹ N-terminal region and the MG6 domains presented upon C4 cleavage by MASP-2 or C1s. This epitope overlaps with the binding site for C2 CCP domains, rationalizing the observed hC4Nb8 inhibition of CP C3 convertase formation and activity. Isothermal titration calorimetry experiments demonstrated that hC4Nb8 binds C4b 105-fold better than native C4. Furthermore, hC4Nb8 inhibited both CP-mediated transport of immune complexes to follicular dendritic cells and C3 deposition on neuronal cell cultures. Together, these studies demonstrate that hC4Nb8 may be useful for analysis and/or therapeutic-inhibition of complement-driven immune response and pathogenesis.
CR3 and Sex Bias in Neutrophil Activation
Previous reports have shown that neutrophils from female mice have an enhanced ability to kill Staphylococcus aureus, resulting in sex-based differences in innate susceptibility to skin infection. In this Top Read, Pokhrel et al. (p. 1593) demonstrated that complement receptor 3 (CR3), C3, and reactive oxygen species (ROS) contribute to the innate sex bias in neutrophil responses to S. aureus. Female mice had higher serum levels of C3, as well as higher expression of CR3 on bone marrow neutrophils (BMN). BMN from female, but not male mice, showed increased killing activity of S. aureus opsonized with female serum, suggesting that the bactericidal capacity of male BMN is limited by CR3 surface expression and cannot be overcome by increasing C3 in the serum. Blockade of CR3 on BMN of both sexes reduced S. aureus killing by female BMN, but not male BMN, further indicating that male BMN bactericidal capacity is CR3 independent. Compared with male BMN, TNF-α primed female BMN produced enhanced ROS levels, which were significantly reduced by blocking CR3. Together, these data suggest CR3, C3, and ROS contribute to the neutrophil-driven innate sex bias in response to S. aureus infection.
FAP-α in Neutrophil Death
Previous studies have demonstrated that nonapoptotic cell death in cytokine-primed human neutrophils use components of the necroptotic death pathway. In this Top Read, Wang et al. (p. 1653) sought to identify additional components of the nonapoptotic cell death pathway in neutrophils. The authors observed that inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α), a serine protease, also blocked CD44-mediated reactive oxygen species production and cell death, but not FAS receptor–mediated apoptosis. In GM-CSF–primed human neutrophils, FAP-α activity was parallel with receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase like (MLKL), and p38 MAPK activation. Interestingly, neutrophils from patients with rheumatoid arthritis underwent GM-CSF–independent necroptosis following CD44 ligation, and this was blocked by either FAP-α or MLKL inhibitors. Thus, this study provides evidence that FAP-α is involved in the activation of NADPH oxidase in nonapoptotic cell death of neutrophils and, therefore, may be a potential therapeutic target to avoid exaggerated neutrophil death leading to tissue damage.
Ascorbic Acid Enhances KIR Demethylation
Killer Ig-like receptors (KIR), expressed on the surface of mature NK cells, require demethylation of promoter regions to allow gene expression. In this Top Read, Wu et al. (p. 1513) demonstrated that ascorbic acid, a catalyst of ten-eleven translocation (TET) enzyme activity and DNA demethylation, increased KIR expression in a dose-dependent manner. Chromatin immunoprecipitation of CD56bright NK cells cultured with ascorbic acid showed enrichment of transcription factors Runx3, TET2, and TET3 within the KIR promoter. Ascorbic acid cultured NK cell lines transduced to overexpress either TET3, or TET3 and Runx3, showed expression of KIR only on cells overexpressing both TET3 and Runx3, demonstrating a direct role for both factors in promoting KIR expression. Although addition of ascorbic acid led to DNA demethylation of the KIR promoter, whole genome methylation analysis of treated NK cells did not show broad epigenetic changes. Indeed, RNA sequencing did not show any differences in transcriptome profiles. Together, these data show that ascorbic acid can specifically enhance DNA demethylation of the KIR promoter in NK cells, leading to increased surface expression.
- Copyright © 2020 by The American Association of Immunologists, Inc.
