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The complement system is a proteolytic cascade triggered by pathogen and danger‐associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell‐intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system. [ABSTRACT FROM AUTHOR]

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases. [ABSTRACT FROM AUTHOR]

One of the hallmarks of type 1 but also type 2 diabetes is pancreatic islet inflammation, associated with altered pancreatic islet function and structure, if unresolved. IL-1ß is a proinflammatory cytokine which detrimentally affects ß-cell function. In the course of diabetes, complement components, including the central complement protein C3, are deregulated. Previously, we reported high C3 expression in human pancreatic islets, with upregulation after IL-1ß treatment. In the current investigation, using primary human and rodent material and CRISPR/Cas9 gene-edited ß-cells deficient in C3, or producing only cytosolic C3 from a noncanonical in-frame start codon, we report a protective effect of C3 against IL-1ß-induced ß-cell death, that is attributed to the cytosolic fraction of C3. Further investigation revealed that intracellular C3 alleviates IL-1ß-induced ß-cell death, by interaction with and inhibition of Fyn-related kinase (FRK), which is involved in the response of ß-cells to cytokines. Furthermore, these data were supported by increased ß-cell death in vivo in a ß-cell-specific C3 knockout mouse. Our data indicate that a functional, cytoprotective association exists between FRK and cytosolic C3. [ABSTRACT FROM AUTHOR]

Introduction: Streptococcus pyogenes (Group A Streptococcus; GAS) is a pathogen that causes over half a million deaths annually worldwide. Human immune cells respond to GAS infection by activating the NLRP3 inflammasome that leads to pro-inflammatory cytokines release which acts to control infection. We investigated the role of C4b-binding protein (C4BP) and Factor H (FH) in the inflammasome response to GAS, as they are recruited by GAS to prevent complement deposition and limit phagocytosis., Methods: Inflammasome response was investigated using isolated primary human cells and the GAS-AP1 strain. Cytokine responses were evaluated by ELISA. C4BP internalisation was investigated using confocal microscopy. Western blotting was used to evaluate the activation of NLRP3 inflammasome components., Results: IL-1β release, induced by GAS-AP1, was inhibited by FH which interferes with priming of human cells. In contrast, C4BP restricted the IL-1β response with no effect on cell priming. C4BP was engulfed by cells together with bacteria and excluded from low-pH vesicles, but localised within the cytosol and near the ASC speck inflammasome complex. C4BP did not inhibit either the inflammasome complex assembly or caspase-1 activation. However, C4BP limited the cleavage of gasderminD N-terminal fragments by interfering with caspase-1 enzymatic activity., Conclusion: Our results provide new insights on the effect of FH and internalised C4BP to control GAS sensing by inflammasomes., (The Author(s). Published by S. Karger AG, Basel.)

The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role in immunological tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGF-beta (TGF-β) and IL-10, while also suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for glioma patients. We confirmed the effect of FH on glioma development in a mouse model, where FH knockdown was associated with decrease in number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (p=0.064). Since the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

Glioblastoma (GBM) is the most aggressive form of glioma with a high rate of relapse despite intensive treatment. Tumor recurrence is tightly linked to radio-resistance, which in turn is associated with hypoxia. Here, we discovered a strong link between hypoxia and local complement signaling using publicly available bulk, single-cell, and spatially resolved transcriptomic data from patients with GBM. Complement component 3 (C3) and the receptor C3AR1 were both associated with aggressive disease and shorter survival in human glioma. In a genetically engineered mouse model of GBM, we found C3 specifically in hypoxic tumor areas. In vitro, we found an oxygen level-dependent increase in C3 and C3AR1 expression in response to hypoxia in several GBM and stromal cell types. C3a induced M2 polarization of cultured microglia and macrophages in a C3aR-dependent fashion. Targeting C3aR using the antagonist SB290157 prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy while reducing the number of M2-polarized macrophages. Our findings establish a strong link between hypoxia and complement pathways in GBM and support a role of hypoxia-induced C3a/C3aR signaling as a contributor to glioma aggressiveness by regulating macrophage polarization.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases., Competing Interests: Author PWHIP was employed by Gyes BV. FSB, PWHIP, and LAT are listed as inventors on a patent application regarding the application of targeted complement inhibition. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, van de Bovenkamp, Dijkstra, Abendstein, Borggreven, Pool, Zuijderduijn, Gstöttner, Gelderman, Damelang, Vidarsson, Blom, Domínguez-Vega, Parren, Sharp and Trouw.)