C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer.

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 C5aR1hi 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.

Avacopan: First Approval.

Avacopan (TAVNEOS™) is a complement 5a receptor (C5aR) antagonist developed by ChemoCentryx for the treatment of autoimmune diseases including anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. The therapeutic effects of avacopan are attributed to the inhibition of C5aR activity on neutrophils, however, the exact mechanism of therapeutic efficacy in patients with ANCA-associated vasculitis has not been established. In September 2021, avacopan received its first approval in Japan for the treatment of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA), the two most common forms of ANCA-associated vasculitis, where it is being commercialized by Kissei Pharmaceutical through a partnership with Vifor Pharma. In October 2021, avacopan was approved in the USA as an adjunctive treatment in adults for severe active ANCA-associated vasculitis (specifically MPA and GPA) in combination with standard therapy including glucocorticoids (avacopan does not eliminate glucocorticoid use). Avacopan has received a positive opinion in the EU, and is also undergoing regulatory review in Switzerland and Canada. Avacopan is being investigated for the treatment of complement component 3 glomerulopathy, hidradenitis suppurativa, lupus nephritis and IgA nephropathy. This article summarizes the milestones in the development of avacopan leading to these first approvals in Japan and the USA.

C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages.

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme ß-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8-10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.

Complement C3a and C5a receptor blockade modulates regulatory T cell conversion in head and neck cancer.

BACKGROUND: Resistance to therapy is a major problem in treating head and neck squamous cell carcinomas (HNSCC). Complement system inhibition has been shown to reduce tumor growth, metastasis, and therapeutic resistance in other tumor models, but has yet to be explored in the context of HNSCC. Here, we tested the effects of complement inhibition and its therapeutic potential in HNSCC. METHODS: We conducted our studies using two Human Papilloma Virus (HPV)-negative HNSCC orthotopic mouse models. Complement C3aR and C5aR1 receptor antagonists were paired with radiation therapy (RT). Tumor growth was measured and immune populations from tumor, lymph node, and peripheral blood were compared among various treatment groups. Genetically engineered mouse models DEREG and C3-/- were used in addition to standard wild type models. Flow cytometry, clinical gene sets, and in vitro assays were used to evaluate the role complement receptor blockade has on the immunological makeup of the tumor microenvironment. RESULTS: In contrast to established literature, inhibition of complement C3a and C5a signaling using receptor antagonists accelerated tumor growth in multiple HNSCC cell lines and corresponded with increased frequency of regulatory T cell (Treg) populations. Local C3a and C5a signaling has importance for CD4 T cell homeostasis and eventual development into effector phenotypes. Interruption of this signaling axis drives a phenotypic conversion of CD4+ T cells into Tregs, characterized by enhanced expression of Foxp3. Depletion of Tregs reversed tumor growth, and combination of Treg depletion and C3a and C5a receptor inhibition decreased tumor growth below that of the control groups. Complete knockout of C3 does not harbor the expected effect on tumor growth, indicating a still undetermined compensatory mechanism. Dexamethasone is frequently prescribed to patients undergoing RT and inhibits complement activation. We report no deleterious effects associated with dexamethasone due to complement inhibition. CONCLUSIONS: Our data establish Tregs as a pro-tumorigenic driver during complement inhibition and provide evidence that targeted C3a and C5a receptor inhibition may add therapeutic advantage when coupled with anti-Treg therapy.

Long-term use and remission of granulomatosis with polyangiitis with the oral C5a receptor inhibitor avacopan.

Granulomatosis with polyangiitis (GPA) is a rare antineutrophil cytoplasm antibody-associated vasculitis. Several therapeutic advances have occurred over the past two decades, but relapse rate remains high and refractory cases are not uncommon. Here, we present the case of a female patient diagnosed with GPA at the age of 9 years with a severe, multirelapsing disease course which failed to adequately respond to conventional therapies. Avacopan, a novel C5a receptor inhibitor, was started based on phase II studies that showed promise as a steroid-sparing adjunct. The patient was able to successfully reduce her glucocorticoid dose and reduce her immunosuppressive treatments without another flare. She has been on avacopan for 35 months, had no adverse events that required its discontinuation, and her disease is in sustained remission.

C5aR deficiency attenuates the breast cancer development via the p38/p21 axis.

Emerging evidence has shown activation of the complement component C5 to C5a in cancer tissues and C5aR expression in breast cancer cells relates to the tumor development and poor prognosis, suggesting the involvement of complement C5a/C5aR pathway in the breast cancer pathogenesis. In this study, we found that as compared to the non-tumoral tissues, both C5aR and MAPK/p38 showed an elevated expression, but p21/p-p21 showed lower expression, in the tumoral tissues of breast cancer patients. Mice deficient in C5aR or mice treated with the C5aR antagonist exhibited attenuation of breast cancer growth and reduction in the p38/p-p38 expression, but increase in p21/p-p21 expression, in the tumor tissues. Pre-treatment of the breast cancer cells with recombinant C5a resulted in reduced p21 expression, and MAPK/p38 inhibitors prevented C5a-induced reduction in p21 expression, suggesting the involvement of the MAPK/p38 signaling pathway in the C5a/C5aR-mediated suppression of p21/p-p21 expression. These results provide evidence that breast cancer development may rely on C5a/C5aR interaction, for which MAPK/p38 pathway participate in down-regulating the p21 expression. Inhibition of C5a/C5aR pathway is expected to be helpful for the treatment of patients with breast cancer.

Pharmacological characterisation of small molecule C5aR1 inhibitors in human cells reveals biased activities for signalling and function.

The complement fragment C5a is a core effector of complement activation. C5a, acting through its major receptor C5aR1, exerts powerful pro-inflammatory and immunomodulatory functions. Dysregulation of the C5a-C5aR1 axis has been implicated in numerous immune disorders, and the therapeutic inhibition of this axis is therefore imperative for the treatment of these diseases. A myriad of small-molecule C5aR1 inhibitors have been developed and independently characterised over the past two decades, however the pharmacological properties of these compounds has been difficult to directly compare due to the wide discrepancies in the model, read-out, ligand dose and instrumentation implemented across individual studies. Here, we performed a systematic characterisation of the most commonly reported and clinically advanced small-molecule C5aR1 inhibitors (peptidic: PMX53, PMX205 and JPE1375; non-peptide: W545011, NDT9513727, DF2593A and CCX168). Through signalling assays measuring C5aR1-mediated cAMP and ERK1/2 signalling, and ß-arrestin 2 recruitment, this study highlighted the signalling-pathway dependence of the rank order of potencies of the C5aR1 inhibitors. Functional experiments performed in primary human macrophages demonstrated the high insurmountable antagonistic potencies for the peptidic inhibitors as compared to the non-peptide compounds. Finally, wash-out studies provided novel insights into the duration of inhibition of the C5aR1 inhibitors, and confirmed the long-lasting antagonistic properties of PMX53 and CCX168. Overall, this study revealed the potent and prolonged antagonistic activities of selected peptidic C5aR1 inhibitors and the unique pharmacological profile of CCX168, which thus represent ideal candidates to fulfil diverse C5aR1 research and clinical therapeutic needs.

Complement 5 Inhibition Ameliorates Hepatic Ischemia/reperfusion Injury in Mice, Dominantly via the C5a-mediated Cascade.

BACKGROUND: Hepatic ischemia/reperfusion injury (IRI) is a serious complication in liver surgeries, including transplantation. Complement activation seems to be closely involved in hepatic IRI; however, no complement-targeted intervention has been clinically applied. We investigated the therapeutic potential of Complement 5 (C5)-targeted regulation in hepatic IRI. METHODS: C5-knockout (B10D2/oSn) and their corresponding wild-type mice (WT, B10D2/nSn) were exposed to 90-minute partial (70%) hepatic ischemia/reperfusion with either anti-mouse-C5 monoclonal antibody (BB5.1) or corresponding control immunoglobulin administration 30 minutes before ischemia. C5a receptor 1 antagonist was also given to WT to identify which cascade, C5a or C5b-9, is dominant. RESULTS: C5-knockout and anti-C5-Ab administration to WT both significantly reduced serum transaminase release and histopathological damages from 2 hours after reperfusion. This improvement was characterized by significantly reduced CD41+ platelet aggregation, maintained F4/80+ cells, and decreased high-mobility group box 1 release. After 6 hours of reperfusion, the infiltration of CD11+ and Ly6-G+ cells, cytokine/chemokine expression, single-stranded DNA+ cells, and cleaved caspase-3 expression were all significantly alleviated by anti-C5-Ab. C5a receptor 1 antagonist was as effective as anti-C5-Ab for reducing transaminases. CONCLUSIONS: Anti-C5 antibody significantly ameliorated hepatic IRI, predominantly via the C5a-mediated cascade, not only by inhibiting platelet aggregation during the early phase but also by attenuating the activation of infiltrating macrophages/neutrophils and hepatocyte apoptosis in the late phase of reperfusion. Given its efficacy, clinical availability, and controllability, C5-targeted intervention may provide a novel therapeutic strategy against hepatic IRI.

Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis.

OBJECTIVE: To provide an alternative therapeutic modality for rheumatoid arthritis (RA), a novel bispecific antibody (BsAb) targeting human tumor necrosis factor α (TNF-α) and human complement component C5a was constructed. RESULTS: BsAb was expressed in Pichia pastoris and secreted into the culture medium as a functional protein. In vitro functional study demonstrated that BsAb could simultaneously bind to TNF-α and C5a and neutralize their biological actions. Furthermore, BsAb showed significant improvements in both the antigen-binding affinity and the neutralizing ability as compared to its original antibodies produced in E. coli. It was also found that TNF-α and C5a had an additive/synergistic effect on promoting the production of inflammatory cytokines and chemokines and C5a receptor (C5aR) expression in human macrophages. Compared to single inhibition of TNF-α or C5a with respective antibody, BsAb showed a superior efficacy in blocking inflammatory cytokines, chemokines, and C5aR response, as well as in lowering the C5a-mediated chemotaxis of macrophages via C5aR in vitro. CONCLUSIONS: With improved production processing and the ability to simultaneously block TNF-α and C5a action, BsAb has a great potential to be developed into a therapeutic agent and may offer a better therapeutic index for RA.

Therapeutic Targeting of Vasculature in the Premetastatic and Metastatic Niches Reduces Lung Metastasis.

In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloid-derived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1ß and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes-based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis.

Inhibition of osteoclast activity by complement regulation with DF3016A, a novel small-molecular-weight C5aR inhibitor.

Recent insights have indicated an active role of the complex complement system not only in immunity, but also in bone remodeling. Evidence from knockout mice and observations from skeletal diseases have drawn attention to the C5a/C5aR axis of the complement cascade in the modulation of osteoclast functions and as potential therapeutic targets for treatment of bone pathologies. With the aim to identify novel C5aR regulators, a medicinal chemistry program was initiated, driven by structural information on a minor pocket of C5aR that has been proposed to be a key motif for C5aR intracellular activation. The impact of the peptidomimetic orthosteric C5aR antagonist (PMX-53), of two newly synthesized allosteric C5aR antagonists (DF2593A, DF3016A), and of C5aR down-regulation by specific siRNAs, were examined for regulation of osteoclastogenesis, using a well-validated in-vitro model starting from RAW264.7 precursor cells. Both pharmacological and molecular approaches reduced osteoclast maturation of RAW264.7 cells induced by receptor-activator of nuclear factor kappa-B ligand (RANKL), which limited the transcription of several differentiation markers evaluated by real-time PCR, including nuclear factor of activated T-cell 1, matrix metalloproteinase-9, cathepsin-K, and tartrate-resistant acid phosphatase. These treatments were ineffective on the subsequent step of osteoclast syncytium formation, apparently as a consequence of reduction of C5aR mRNA levels in the course of osteoclastogenesis, as monitored by real-time PCR. Among the C5aR antagonists analyzed, DF3016A inhibited osteoclast degradation activity through inhibition of C5aR signal transduction and transcription. These data confirm the preclinical relevance of this novel therapeutic candidate.

High Concentration of C5a-Induced Mitochondria-Dependent Apoptosis in Murine Kidney Endothelial Cells.

Patients with a relapse of idiopathic nephrotic syndrome have significantly increased levels of serum complement component 5a (C5a), and proteinuria has been noted in mice treated with C5a via changes in permeability of kidney endothelial cells (KECs) in established animal models. However, the apoptosis of KECs treated with high concentrations of C5a has also been observed. As mitochondrial damage is known to be important in cell apoptosis, the aim of this study was to examine the association between C5a-induced mouse KEC apoptosis and mitochondrial damage. Mouse KECs were isolated and treated with different concentrations of C5a. Cell viability assays showed that a high-concentration mouse recombinant protein C5a (rmC5a) treatment reduced mouse KEC growth. Cell cycle phase analysis, including apoptosis (sub-G1 phase) showed an increased percentage of the subG1 phase with a high-concentration rmC5a treatment. Cytochrome c and caspase 3/9 activities were significantly induced in the mouse KECs after a high-dose rmC5a (50 ng/mL) treatment, and this was rescued by pretreatment with the C5a receptor (C5aR) inhibitor (W-54011) and N-acetylcysteine (NAC). Reactive oxygen species (ROS) formation was detected in C5a-treated mouse KECs; however, W-54011 or NAC pretreatment inhibited high-dose rmC5a-induced ROS formation and also reduced cytochrome c release, apoptotic cell formation, and apoptotic DNA fragmentation. These factors determined the apoptosis of mouse KECs treated with high-dose C5a through C5aR and subsequently led to apoptosis via ROS regeneration and cytochrome c release. The results showed that high concentrations of C5a induced mouse KEC apoptosis via a C5aR/ROS/mitochondria-dependent pathway. These findings may shed light on the potential mechanism of glomerular sclerosis, a process in idiopathic nephrotic syndrome causing renal function impairment.

N-Acetylcysteine Attenuates Cisplatin-Induced Acute Kidney Injury by Inhibiting the C5a Receptor.

N-acetylcysteine has been widely used as a nutritional supplement and drug in humans for its antioxidant properties. The complement activation fragment C5a is a strong proinflammatory molecule that mediates cell adhesion, chemotaxis, and the complex biological functions. However, the effect of NAC on the C5a, and the relationship of those two with cisplatin-induced acute kidney injury are unknown. In cisplatin induced AKI mouse model, mice with NAC administration had a marked improvement in renal function (BUN and Cr), decreased pathological damage, reduced inflammation, and alleviated renal oxidative stress. Furthermore, C5a and C5aR expression in the cisplatin-treated group was notably increased compared with the control group, and this increase could be significantly inhibited by NAC. In addition, neutrophils coexpressed distinctly with C5aR, and the number of infiltrating neutrophils (MPO+ly6G+) and inflammatory factors decreased with NAC treatment in the cisplatin-treated group. Overall, these data demonstrate that NAC could ameliorate cisplatin-induced nephrotoxicity in mice and the protective effects may be conducted by inhibiting the activation of kidney inflammation and the complement system.

LukS-PV induces apoptosis in acute myeloid leukemia cells mediated by C5a receptor.

LukS-PV is one of the two components of Panton-Valentine leucocidin (PVL). Our previous study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) THP-1 and HL-60 cells. C5aR (C5a receptor) is the receptor for PVL, but whether C5aR plays a key role in LukS-PV induced apoptosis is unclear. The aim of this study was to establish whether C5aR plays a physiological role in apoptosis of leukemia cells induced by LukS-PV. We investigated the role of C5aR in leukemia cell apoptosis induced by LukS-PV by pretreatment of THP-1 and HL-60 cells with C5aR antagonist and transfection to knockdown C5aR in THP-1 cells or overexpress C5aR in Jurkat cells before treatment with LukS-PV. Cell apoptosis was analyzed by staining with Annexin V/propidium iodide or Annexin V-PE/7-AAD. Mitochondrial membrane potential (MMP) was determined using JC-1 dye. The expression of apoptosis-associated genes and proteins was identified by qRT-polymerase chain reaction and Western blotting analysis, respectively. As the C5aR antagonist concentration increased, the rate of apoptosis induced by LukS-PV decreased, the MMP increased, and expression of pro-apoptotic Bax and Bak genes and proteins was downregulated while that of anti-apoptotic Bcl-2 and Bcl-x genes and proteins was upregulated. Knockdown of C5aR also decreased LukS-PV-induced THP-1 cell apoptosis. LukS-PV did not induce apoptosis of Jurkat cells, which have no endogenous C5aR expression; however, LukS-PV did induce apoptosis in Jurkat cells after overexpression of C5aR. Correspondingly, the MMP decreased and Bax and Bak were upregulated while Bcl-2 and Bcl-x were downregulated. LukS-PV can induce apoptosis in AML cells by targeting C5aR. C5aR may be a potential therapeutic target for AML and LukS-PV is a candidate targeted drug for the treatment of AML.

Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus with a crude mortality rate of ~35%. Previously, we established a human DPP4 transgenic (hDPP4-Tg) mouse model in which we studied complement overactivation-induced immunopathogenesis. Here, to better understand the pathogenesis of MERS-CoV, we studied the role of pyroptosis in THP-1 cells and hDPP4 Tg mice with MERS-CoV infection. We found that MERS-CoV infection induced pyroptosis and over-activation of complement in human macrophages. The hDPP4-Tg mice infected with MERS-CoV overexpressed caspase-1 in the spleen and showed high IL-1ß levels in serum, suggesting that pyroptosis occurred after infection. However, when the C5a-C5aR1 axis was blocked by an anti-C5aR1 antibody (Ab), expression of caspase-1 and IL-1ß fell. These data indicate that MERS-CoV infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. Pyroptosis and inflammation were suppressed by inhibiting C5aR1. These results will further our understanding of the pathogenesis of MERS-CoV infection.

[What's new in dermatological treatment?] / Quoi de neuf en thérapeutique dermatologique?

The therapeutic revolution in the management of inflammatory dermatoses is under way. The therapeutic arsenal is expanding in the field of psoriasis, including biologics (TNF blockers, anti-IL12/IL23, anti-IL17, and anti-IL23 antibodies), new small molecules (tyrosine kinase inhibitor), and a new biologic for generalized pustular psoriasis (anti-IL36 receptor). New biologics will be soon available in the field of atopic dermatitis in addition to anti-IL4/IL13 antibodies. New targeted treatments of pruritus are also coming (biologics and small molecules). A first randomized placebo-controlled trial has confirmed the interest of JAK inhibitors in alopecia areata. These molecules seem to be also promising in dermatomyositis. Another therapeutic revolution will be technological with the development of new therapeutic agents: small interfering RNA. Recent clinical trials confirmed their efficacy in hereditary amyloidosis.

C5aR antagonist inhibits occurrence and progression of complement C5a induced inflammatory response of microglial cells through activating p38MAPK and ERK1/2 signaling pathway.

OBJECTIVE: To discuss the effect of complement C52 (C5a) and complement C5a receptor (C5aR) antagonists on inflammatory status of mouse microglial cells. MATERIALS AND METHODS: Primary culture was performed on mouse microglial cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect effect of C5a and C5aR antagonists on vitality of microglial cells. The effect of C5a and C5aR antagonists on mRNA expression of p38MAPK and ERK1/2 was determined using quantitative PCR (qPCR). Enzyme linked immunosorbent assay (ELISA) was used to measure expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cells. RESULTS: According to quantitative fluorescent PCR, relative expression of p38MAPK and ERK1/2 mRNA in C5a antagonist treatment group was significantly higher compared to normal group and C5a+C5aR antagonist treatment group (p<0.05). However, the relative expression of the C5a+C5aR antagonist treatment group was significantly lower compared to that of the normal group (p<0.05). Expression of Iba1, p-p38MAPK and p-ERK1/2 proteins in C5a antagonist treatment group was significantly higher than normal group, in C5a+C5aR antagonist treatment group was lower than C5a antagonist treatment group (p<0.05). There were significant differences for IL-6 and TNF-α levels among 5 groups (p<0.05). Expression of cytokines was the highest in 100 nM C5a antagonist treatments and lowest in normal group. CONCLUSIONS: Complement C5a upregulated expression of inflammatory factors in mouse microglial cells, while C5aR antagonist inhibited occurrence and progression of inflammatory status. This was achieved by affecting transcriptional and translational processes of different factors in p38MAPK and ERK1/2 signaling pathway.

CD177-mediated nanoparticle targeting of human and mouse neutrophils.

Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70-90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor.

Blockade of the C5a-C5aR axis alleviates lung damage in hDPP4-transgenic mice infected with MERS-CoV.

The pathogenesis of highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) remains poorly understood. In a previous study, we established an hDPP4-transgenic (hDPP4-Tg) mouse model in which MERS-CoV infection causes severe acute respiratory failure and high mortality accompanied by an elevated secretion of cytokines and chemokines. Since excessive complement activation is an important factor that contributes to acute lung injury after viral infection, in this study, we investigated the role of complement in MERS-CoV-induced lung damage. Our study showed that complement was excessively activated in MERS-CoV-infected hDPP4-Tg mice through observations of increased concentrations of the C5a and C5b-9 complement activation products in sera and lung tissues, respectively. Interestingly, blocking C5a production by targeting its receptor, C5aR, alleviated lung and spleen tissue damage and reduced inflammatory responses. More importantly, anti-C5aR antibody treatment led to decreased viral replication in lung tissues. Furthermore, compared with the sham treatment control, apoptosis of splenic cells was less pronounced in the splenic white pulp of treated mice, and greater number of proliferating splenic cells, particularly in the red pulp, was observed. These data indicate that (1) dysregulated host immune responses contribute to the severe outcome of MERS; (2) excessive complement activation, triggered by MERS-CoV infection, promote such dysregulation; and (3) blockade of the C5a-C5aR axis lead to the decreased tissue damage induced by MERS-CoV infection, as manifested by reduced apoptosis and T cell regeneration in the spleen. Therefore, the results of this study suggest a new strategy for clinical intervention and adjunctive treatment in MERS-CoV cases.

Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration.

BACKGROUND: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration. METHODS: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control. RESULTS: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1. CONCLUSIONS: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.