PMX-53 as a Dual CD88 antagonist and an Agonist for Mas-Related Gene 2 (MrgX2) in Human Mast Cells

Human mast cells express the G protein coupled receptor (GPCR) for C5a (CD88). Previous studies indicated that C5a could cause mast cell degranulation, at least in part, via a mechanism similar to that proposed for basic neuropeptides such as substance P, possibly involving Mas-related gene 2 (MrgX2). We therefore sought to more clearly define the receptor specificity for C5a-induced mast cell degranulation. We found that LAD2, a human mast cell line, and CD34+ cell-derived primary mast cells express functional MrgX1 and MrgX2 but the immature human mast cell line HMC-1 does not. A potent CD88 antagonist, PMX-53 (10 nM) inhibited C5ainduced Ca2+ mobilization in HMC-1 cells, but at higher concentrations (≥30 nM) it caused degranulation in LAD2 mast cells, CD34+ cell-derived mast cells, and RBL-2H3 cells stably expressing MrgX2. PMX-53 did not, however, activate RBL-2H3 cells expressing MrgX1. Although C5a induced degranulation in LAD2 and CD34+ cell-derived mast cells, it did not activate RBL-2H3 cells expressing MrgX1 or MrgX2. Replacement of Trp with Ala and Arg with dArg abolished the ability of PMX-53 to inhibit C5a-induced Ca2+ mobilization in HMC-1 cells and to cause degranulation in RBL-2H3 cells expressing MrgX2. These findings demonstrate that C5a does not use MrgX1 or MrgX2 for mast cell degranulation. Moreover, it reveals the novel finding that PMX-53 functions as a potent CD88 antagonist and a low-affinity agonist for MrgX2. Furthermore, Trp and Arg residues are required for the ability of PMX53 to act as both a CD88 antagonist and a MrgX2 agonist. Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics

From Orphan Drugs to Adopted Therapies: Advancing C3-Targeted Intervention to the Clinical Stage

Complement dysregulation is increasingly recognized as an important pathogenic driver in a number of clinical disorders. Complement-triggered pathways intertwine with key inflammatory and tissue destructive processes that can either increase the risk of disease or exacerbate pathology in acute or chronic conditions. The launch of the first complement-targeted drugs in the clinic has undeniably stirred the field of complement therapeutic design, providing new insights into complement\u27s contribution to disease pathogenesis and also helping to leverage a more personalized, comprehensive approach to patient management. In this regard, a rapidly expanding toolbox of complement therapeutics is being developed to address unmet clinical needs in several immune-mediated and inflammatory diseases. Elegant approaches employing both surface-directed and fluid-phase inhibitors have exploited diverse components of the complement cascade as putative points of therapeutic intervention. Targeting C3, the central hub of the system, has proven to be a promising strategy for developing biologics as well as small-molecule inhibitors with clinical potential. Complement modulation at the level of C3 has recently shown promise in preclinical primate models, opening up new avenues for therapeutic intervention in both acute and chronic indications fueled by uncontrolled C3 turnover. This review highlights recent developments in the field of complement therapeutics, focusing on C3-directed inhibitors and alternative pathway (AP) regulator-based approaches. Translational perspectives and considerations are discussed, particularly with regard to the structure-guided drug optimization and clinical advancement of a new generation of C3-targeted peptidic inhibitors

Complementing the cancer-immunity cycle

Reactivation of cytotoxic CD8+ T-cell responses has set a new direction for cancer immunotherapy. Neutralizing antibodies targeting immune checkpoint programmed cell death protein 1 (PD-1) or its ligand (PD-L1) have been particularly successful for tumor types with limited therapeutic options such as melanoma and lung cancer. However, reactivation of T cells is only one step toward tumor elimination, and a substantial fraction of patients fails to respond to these therapies. In this context, combination therapies targeting more than one of the steps of the cancer-immune cycle may provide significant benefits. To find the best combinations, it is of upmost importance to understand the interplay between cancer cells and all the components of the immune response. This review focuses on the elements of the complement system that come into play in the cancer-immunity cycle. The complement system, an essential part of innate immunity, has emerged as a major regulator of cancer immunity. Complement effectors such as C1q, anaphylatoxins C3a and C5a, and their receptors C3aR and C5aR1, have been associated with tolerogenic cell death and inhibition of antitumor T-cell responses through the recruitment and/or activation of immunosuppressive cell subpopulations such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or M2 tumor-associated macrophages (TAMs). Evidence is provided to support the idea that complement blocks many of the effector routes associated with the cancer-immunity cycle, providing the rationale for new therapeutic combinations aimed to enhance the antitumor efficacy of anti-PD-1/PD-L1 checkpoint inhibitors

Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy.

Cancer immunotherapy relies upon the ability of T cells to infiltrate tumors. The endothelium constitutes a barrier between the tumor and effector T cells, and the ability to manipulate local vascular permeability could be translated into effective immunotherapy. Here, we show that in the context of adoptive T cell therapy, antitumor T cells, delivered at high enough doses, can overcome the endothelial barrier and infiltrate tumors, a process that requires local production of C3, complement activation on tumor endothelium and release of C5a. C5a, in turn, acts on endothelial cells promoting the upregulation of adhesion molecules and T-cell homing. Genetic deletion of C3 or the C5a receptor 1 (C5aR1), and pharmacological blockade of C5aR1, impaired the ability of T cells to overcome the endothelial barrier, infiltrate tumors, and control tumor progression in vivo, while genetic chimera mice demonstrated that C3 and C5aR1 expression by tumor stroma, and not leukocytes, governs T cell homing, acting on the local endothelium. In vitro, endothelial C3 and C5a expressions were required for endothelial activation by type 1 cytokines. Our data indicate that effective immunotherapy is a consequence of successful homing of T cells in response to local complement activation, which disrupts the tumor endothelial barrier

Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae

Protection of innate immunity by C5aR antagonist in septic mice

Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125Iâ C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5aâ exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5aâ induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.â Huberâ Lang, M. S., Riedeman, N. C., Sarma, J. V., Younkin, E. M., McGuire, S. R., Laudes, I. J., Lu, K. T., Guo, R.â F., Neff, T. A., Padgaonkar, V. A., Lambris, J. D., Spruce, L., Mastellos, D., Zetoune, F. S., Ward, P. A. Protection of innate immunity by C5aR antagonist in septic mice. FASEB J. 16, 1567â 1574 (2002)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154360/1/fsb2fj020209com.pd

Complement-Dependent Mechanisms and Interventions in Periodontal Disease

Periodontitis is a prevalent inflammatory disease that leads to the destruction of the tooth-supporting tissues. Current therapies are not effective for all patients and this oral disease continues to be a significant public health and economic burden. Central to periodontal disease pathogenesis is a reciprocally reinforced interplay between microbial dysbiosis and destructive inflammation, suggesting the potential relevance of host-modulation therapies. This review summarizes and discusses clinical observations and pre-clinical intervention studies that collectively suggest that complement is hyperactivated in periodontitis and that its inhibition provides a therapeutic benefit. Specifically, interception of the complement cascade at its central component, C3, using a locally administered small peptidic compound (Cp40/AMY-101) protected non-human primates from induced or naturally occurring periodontitis. These studies indicate that C3-targeted intervention merits investigation as an adjunctive treatment of periodontal disease in humans

Microbes Bind Complement Inhibitor Factor H via a Common Site

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19–20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens viautilization of a “superevasion site.

Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β2-glycoprotein I and interferes with innate immune recognition