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The crystal structure of the complement receptor C5aR1 bound to a small-molecule antagonist provides insights into this receptor class and how they could be better targeted therapeutically. Structure of a complement receptor The complement system plays a central part in innate immune responses and provides a basic defence mechanism against infection. C5a is one component that is produced on activation of this system and is an inflammatory mediator that interacts with the G-protein-coupled receptor (GPCR) C5aR1. As such, C5aR1 inhibitors are anti-inflammatory targets for the treatment of sepsis, psoriasis and rheumatoid arthritis. Fiona Marshall and colleagues report the crystal structure of the complement C5a receptor bound to a small-molecule antagonist. These findings offer molecular-level insights into this class of receptors and how they could be better targeted therapeutically. As with some other recently reported GPCR structures, the ligand binds to an allosteric binding pocket outside the transmembrane bundle. This work also explains the difference in pharmacology between human and rodent receptors. The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin 1 , 2 . Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn’s disease and ischaemia-reperfusion injuries 1 . More recently, a role of C5a in neurodegenerative conditions such as Alzheimer’s disease has been identified 3 . Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 5 and NDT9513727 6 , have been identified by C5a radioligand-binding assays 4 . NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp213 5.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell–dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR–CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection. Heeger and colleagues report that activated B cells dynamically regulate the expression of complement regulatory proteins via the transcription factor BCL6. C3 convertase activity and C3aR1–C5aR1 signaling were both necessary for optimal B cell activation and germinal center formation.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1 −/− mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo. In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4. As more intersection points between platelets and the immune system are found, the role of platelets for vessel growth in the adult organism remains unclear. The authors demonstrate that platelets negatively modulate revascularization through CXCL4 secretion induced by activation C5aR1 on their surface.

Complement fragment (C)5a is a 74 residue pro‐inflammatory polypeptide produced during activation of the complement cascade of serum proteins in response to foreign surfaces such as microorganisms and tissue damaged by physical or chemical injury. C5a binds to at least two seven‐transmembrane domain receptors, C5aR (C5R1, CD88) and C5L2 (gpr77), expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages, neutrophils and T cells. C5aR is a classical G protein‐coupled receptor that signals through Gαi and Gα16, whereas C5L2 does not appear to couple to G proteins and has no known signalling activity. Although C5a was first described as an anaphylatoxin and later as a leukocyte chemoattractant, the widespread expression of C5aR suggested more general functionality. Our understanding of the physiology of C5a has improved significantly in recent years through exploitation of receptor knockout and knockin mice, C5 and C5a antibodies, soluble recombinant C5a and C5a analogues and newly developed receptor antagonists. C5a is now also implicated in non‐immunological functions associated with developmental biology, CNS development and neurodegeneration, tissue regeneration, and haematopoiesis. Combined receptor mutagenesis, molecular modelling, structure‐activity relationship studies and species dependence for ligand potency on C5aR have been helpful for identifying ligand binding sites on the receptor and for defining mechanisms of receptor activation and inactivation. This review will highlight major developments in C5a receptor research that support C5aR as an important therapeutic target. The intriguing possibilities raised by the existence of a non‐signalling C5a receptor are also discussed. British Journal of Pharmacology (2007) 152, 429–448; doi:10.1038/sj.bjp.0707332; published online 2 July 2007

Alzheimer’s disease (AD) is the leading cause of dementia in older adults, and the need for effective, sustainable therapeutic targets is imperative. The complement pathway has been proposed as a therapeutic target. C5aR1 inhibition reduces plaque load, gliosis, and memory deficits in animal models, however, the cellular bases underlying this neuroprotection were unclear. Here, we show that the C5aR1 antagonist PMX205 improves outcomes in the Arctic48 mouse model of AD. A combination of single cell and single nucleus RNA-seq analysis of hippocampi derived from males and females identified neurotoxic disease-associated microglia clusters in Arctic mice that are C5aR1-dependent, while microglial genes associated with synapse organization and transmission and learning were overrepresented in PMX205-treated mice. PMX205 also reduced neurotoxic astrocyte gene expression, but clusters associated with protective responses to injury were unchanged. C5aR1 inhibition promoted mRNA-predicted signaling pathways between brain cell types associated with cell growth and repair, while suppressing inflammatory pathways. Finally, although hippocampal plaque load was unaffected, PMX205 prevented deficits in short-term memory in female Arctic mice. In conclusion, C5aR1 inhibition prevents cognitive loss, limits detrimental glial polarization while permitting neuroprotective responses, as well as leaving most protective functions of complement intact, making C5aR1 antagonism an attractive therapeutic strategy for AD. The complement pathway has been proposed as a therapeutic target for Alzheimer’s disease (AD). Pharmacologic inhibition of C5aR1 suppresses disease-enhancing processes and promotes disease mitigating pathways in an aggressive model of AD.

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases, including BRCA1/2 mutant breast cancer, responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing, we identify a TAM_C3 cluster, expressing genes implicated in anti-inflammatory activity, that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1 hi cells increases and decreases PARPi sensitivity, respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus, targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies. PARP inhibitors (PARPi) have been approved for the treatment of metastatic triple-negative breast cancer (BC), however resistance and recurrence are often observed. Here, in preclinical models of BRCA1/2 wild type and homologous recombination competent BC, the authors show that C5aR1-positive tumor associated macrophages are associated with PARPi-resistance, suggesting targeting C5aR1 as a therapeutic option.

Background The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer’s disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation. Methods To elucidate the effects of C5a–C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7–10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states. Results ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a–C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a–C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers. Conclusion C5a–C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.

Neutrophil recruitment into the joint is a hallmark of inflammatory arthritides, including rheumatoid arthritis (RA). In a mouse model of autoantibody-induced inflammatory arthritis, neutrophils infiltrate the joint via multiple chemoattractant receptors, including the leukotriene B ₄ (LTB ₄) receptor BLT1 and the chemokine receptors CCR1 and CXCR2. Once in the joint, neutrophils perpetuate their own recruitment by releasing LTB ₄ and IL-1β, presumably after activation by immune complexes deposited on joint structures. Two pathways by which immune complexes may activate neutrophils include complement fixation, resulting in the generation of C5a, and direct engagement of Fcγ receptors (FcγRs). Previous investigations showed that this model of autoantibody-induced arthritis requires the C5a receptor C5aR and FcγRs, but the simultaneous necessity for both pathways was not understood. Here we show that C5aR and FcγRs work in sequence to initiate and sustain neutrophil recruitment in vivo. Specifically, C5aR activation of neutrophils is required for LTB ₄ release and early neutrophil recruitment into the joint, whereas FcγR engagement upon neutrophils induces IL-1β release and subsequent neutrophil-active chemokine production, ensuring continued inflammation. These findings support the concept that immune complex-mediated leukocyte activation is not composed of overlapping and redundant pathways, but that each element serves a distinct and critical function in vivo, culminating in tissue inflammation.

Over 60% of women with endometriosis experience abdominopelvic pain and broader pain manifestations, including chronic back pain, fibromyalgia, chronic fatigue, vulvodynia, and migraine. Although the imbalance of proinflammatory mediators, including the complement component C5a, is associated with endometriosis-related pain, the mechanisms causing widespread pain and the C5a role remain unclear. Female mice and women with endometriosis exhibit increased plasma C5a levels and pain. We hypothesize the Schwann cells involvement in endometriotic pain. Here, we show that silencing the C5a receptor (C5aR1) in Schwann cells blocks the C5a-induced activation of the NLRP1 inflammasome and subsequent release of interleukin-1β (IL-1β). Macrophages, recruited to sciatic/trigeminal nerves by IL-1β from Schwann cells, increase oxidative stress, which activates the proalgesic TRPA1 pathway, resulting in widespread pain. These findings reveal a pathway involving Schwann cell C5aR1, NLRP1/IL-1β activation, macrophage recruitment, oxidative stress, and TRPA1 engagement, contributing to pain in a mouse model of endometriosis. Endometriosis affects over 60% of women, leading to widespread pain. Here, the authors show that blocking C5a receptor (C5aR1) in Schwann cells reduces pain by inhibiting pathways that trigger inflammation, oxidative stress, and nerve sensitivity, revealing a potential therapeutic target in endometriosis pain management.