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Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches. Properdin is the only known positive regulator of the human complement system, stabilising the convertase C3 in the alternative pathway of complement activation. Here, the authors report the identification and characterisation of a species-specific properdin inhibitor CirpA, derived from tick saliva.

Aberrant complement activation can cause damage to newly formed fetal-derived structures and excessive inflammatory response at the feto-maternal interface, contributing to pregnancy-related complications, including preeclampsia (PE), which is one of the most severe pathologies in new-borns. Properdin is the only known positive regulator of the complement alternative pathway, as it stabilizes the inherently labile C3bBb complex and amplifies its activity. This study describes the presence of properdin in PE and investigates its role in the pathogenesis. We examined the distribution and expression of properdin at both the transcript and protein levels in term placental tissue, serum, placental syncytiotrophoblast microvesicles (STBMs), and circulating placental exosomes from PE women compared to healthy mothers, using RT-qPCR, western blot, immunohistochemistry, transmission electron microscopy (TEM), and immunofluorescence. To link properdin levels with alternative pathway complement factors, we also assessed the expression of C3 and C5. PE placentae showed significantly higher properdin, C3 and C5 at transcript as well as protein levels compared to healthy placentae. Conversely, properdin levels in serum, STBMs, and circulating placental exosomes were lower in PE compared to healthy pregnancies. Immunohistochemical analysis revealed properdin distribution throughout the PE placentae, with higher concentrations at the syncytial knots containing pyknotic nuclei were observed via TEM, along with elevated levels of cleaved caspase 3. Thus, properdin was significantly upregulated in the PE placentae, along with C3 and C5, and might be associated with the apoptotic nuclei inside syncytial knots. This evidence suggests that properdin may trigger complement-mediated damage to the placental barrier, exacerbating the development of PE placentae.

Properdin and factor H (FH), the two regulatory proteins of the alternative complement pathway, oppose each other to maintain the complement system's activation. While properdin upregulates, FH downregulates the complement alternative pathway. The current study evaluated the expression of properdin and FH transcripts and proteins in the placental tissues and umbilical cords (UC) of preeclampsia (PE), gestational diabetes mellitus (GDM), and recurrent pregnancy loss (RPL) compared to normal healthy pregnancy (N). The tissue histology of PE, GDM and RPL were observed using haematoxylin-eosin and Masson's trichrome staining. To understand the expression and distribution of properdin and FH, RT-qPCR, western blot, and immunohistochemistry were carried out. The expressions of two additional complement components, C3 and C5, were also detected by western blot. The placentae from PE and GDM showed substantial collagen and fibrinoid deposition, thicker foetal blood capillaries, and a considerable number of syncytial knots. There was a significant rise in the level of properdin and significant decline in the level of FH at both mRNA and protein levels in the placentae and umbilical cord of PE compared to N; in GDM placentae, both properdin and FH were significantly elevated compared to N. In the case of RPL placentae, similar to PE, properdin expression was high while FH expression level was low. In both PE and RPL placentae, C3 and C5 levels were high, suggesting possibility of overactivation of complement proteins in the placenta. The observed elevated properdin level can contribute to the heightened inflammatory response in PE, GDM and RPL placentae. Low FH and high C3 and C5 in the placenta possibly suggests dysregulated complement activation in PE and RPL.

Intestinal ischemia reperfusion (IR)-induced tissue injury represents an acute inflammatory response with significant morbidity and mortality. The mechanism of IR-induced injury is not fully elucidated, but recent studies suggest a critical role for complement activation and for differences between sexes. To test the hypothesis that complement initiation differs by sex in intestinal IR, we performed intestinal IR on male and female WT C57B6L/, C1q -/- , MBL -/- , or properdin (P) -/- mice. Intestinal injury, C3b and C5a production and ex vivo secretions were analyzed. Initial studies demonstrated a difference in complement mRNA and protein in male and female WT mice. In response to IR, male C1q-, MBL- and P-deficient mice sustained less injury than male WT mice. In contrast, only female MBL -/- mice sustained significantly less injury than female wildtype mice. Importantly, wildtype, C1q -/- and P -/- female mice sustained significant less injury than the corresponding male mice. In addition, both C1q and MBL expression and deposition increased in WT male mice, while only elevated MBL expression and deposition occurred in WT female mice. These data suggested that males use both C1q and MBL pathways, while females tend to depend on lectin pathway during intestinal IR. Females produced significantly less serum C5a in MBL -/- and P -/- mice. Our findings suggested that complement activation plays a critical role in intestinal IR in a sex-dependent manner.

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia–reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (P KO ) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by P KO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H 2 O 2 -increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H 2 O 2 but inhibited by P KO . These results were confirmed by counting phagocytosed H 2 O 2 -induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, P KO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Phagocytosis plays vital roles in injury and repair, while its regulation by properdin and innate repair receptor, a heterodimer receptor of erythropoietin receptor (EPOR)/β common receptor (βcR), in renal ischaemia-reperfusion (IR) remains unclear. Properdin, a pattern recognition molecule, facilitates phagocytosis by opsonizing damaged cells. Our previous study showed that the phagocytic function of tubular epithelial cells isolated from properdin knockout ( P KO ) mouse kidneys was compromised, with upregulated EPOR in IR kidneys that was further raised by P KO at repair phase. Here, helix B surface peptide (HBSP), derived from EPO only recognizing EPOR/βcR, ameliorated IR-induced functional and structural damage in both P KO and wild-type (WT) mice. In particular, HBSP treatment led to less cell apoptosis and F4/80+ macrophage infiltration in the interstitium of P KO IR kidneys compared to the WT control. In addition, the expression of EPOR/βcR was increased by IR in WT kidneys, and furthered increased in IR P KO kidneys, but greatly reduced by HBSP in the IR kidneys of P KO mice. HBSP also increased PCNA expression in IR kidneys of both genotypes. Moreover, iridium-labelled HBSP (HBSP-Ir) was localized mainly in the tubular epithelia after 17-h renal IR in WT mice. HBSP-Ir also anchored to mouse kidney epithelial (TCMK-1) cells treated by H 2 O 2 . Both EPOR and EPOR/βcR were significantly increased by H 2 O 2 treatment, while further increased EPOR was showed in cells transfected with small interfering RNA (siRNA) targeting properdin, but a lower level of EPOR was seen in EPOR siRNA and HBSP-treated cells. The number of early apoptotic cells was increased by EPOR siRNA in H 2 O 2 -treated TCMK-1, but markedly reversed by HBSP. The phagocytic function of TCMK-1 cells assessed by uptake fluorescence-labelled E.coli was enhanced by HBSP dose-dependently. Our data demonstrate for the first time that HBSP improves the phagocytic function of tubular epithelial cells and kidney repair post IR injury, via upregulated EPOR/βcR triggered by both IR and properdin deficiency.

C3 glomerulopathies (C3G) are ultra-rare complement-mediated diseases that lead to end-stage renal disease (ESRD) within 10 years of diagnosis in ~50% of patients. Overactivation of the alternative pathway (AP) of complement in the fluid phase and on the surface of the glomerular endothelial glycomatrix is the underlying cause of C3G. Although there are animal models for C3G that focus on genetic drivers of disease, in vivo studies of the impact of acquired drivers are not yet possible. Here we present an in vitro model of AP activation and regulation on a glycomatrix surface. We use an extracellular matrix substitute (MaxGel) as a base upon which we reconstitute AP C3 convertase. We validated this method using properdin and Factor H (FH) and then assessed the effects of genetic and acquired drivers of C3G on C3 convertase. We show that C3 convertase readily forms on MaxGel and that this formation was positively regulated by properdin and negatively regulated by FH. Additionally, Factor B (FB) and FH mutants impaired complement regulation when compared to wild type counterparts. We also show the effects of C3 nephritic factors (C3Nefs) on convertase stability over time and provide evidence for a novel mechanism of C3Nef-mediated C3G pathogenesis. We conclude that this ECM-based model of C3G offers a replicable method by which to evaluate the variable activity of the complement system in C3G, thereby offering an improved understanding of the different factors driving this disease process.

The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface–bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement–mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement–mediated injury and support its therapeutic targeting in complement-dependent human diseases.

Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.

Background and Objectives: Tumours are often low immunogenic. The role of complement, an innate immune defence system, in tumour control has begun to be elucidated, but findings are conflicting. A role for properdin, an amplifier of complement activation, in tumour control has recently been implicated. Materials and Methods: Properdin-deficient and congenic wildtype mice were injected subcutaneously with B16F10 melanoma cells. Tumour mass and chemokine profile were assessed. The frequencies of CD45+CD11b+ Gr-1+ cells were determined from tumours and spleens, and CD206+ F4/80+ cells were evaluated in spleens. Sera were analysed for C5a, sC5b-9, and CCL2. Results: Whilst there was no difference in tumour growth at study endpoint, properdin-deficient mice had significantly fewer myeloid-derived suppressor cells (MDSCs) in their tumours and spleens. Splenic M2 type macrophages and serum levels of C5a, sC5b-9, and CCL2 were decreased in properdin-deficient compared to wildtype mice. Conclusions: The presence of intact complement amplification sustains an environment that lessens potential anti-tumour responses.