C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Complement system is overactivated in patients with IgA nephropathy after COVID-19.

IgA nephropathy (IgAN), which has been confirmed as a complement mediated autoimmune disease, is also one form of glomerulonephritis associated with COVID-19. Here, we aim to investigate the clinical and immunological characteristics of patients with IgAN after COVID-19. The level of plasma level of C5a (p < 0.001), soluble C5b-9 (p = 0.018), FHR5 (p < 0.001) were all significantly higher in Group CoV (33 patients with renal biopsy-proven IgAN experienced COVID-19) compared with Group non-CoV (44 patients with IgAN without COVID-19), respectively. Compared with Group non-CoV, the intensity of glomerular C4d (p = 0.017) and MAC deposition (p < 0.001) and Gd-IgA1 deposition (p = 0.005) were much stronger in Group CoV. Our finding revealed that for IgAN after COVID-19, mucosal immune responses to SARS-CoV-2 infection may result in the overactivation of systemic and renal local complement system, and increased glomerular deposition of Gd-IgA1, which may lead to renal dysfunction and promote renal progression in IgAN patients.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers.

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. STATEMENT OF SIGNIFICANCE: Chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy. We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ T cell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.

Role of the Intracellular Sodium Homeostasis in Chemotaxis of Activated Murine Neutrophils.

The importance of the intracellular Ca2+ concentration ([Ca2+]i) in neutrophil function has been intensely studied. However, the role of the intracellular Na+ concentration ([Na+]i) which is closely linked to the intracellular Ca2+ regulation has been largely overlooked. The [Na+]i is regulated by Na+ transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+]i. fMLF induces a sustained increase of [Na+]i, surprisingly, reaching higher values in TRPM2-/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+]i. The difference in [Na+]i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na+ concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2-/- neutrophils. Increasing the [Na+]i by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na+ homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na+ concentration gradients. Increasing the [Na+]i by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.

Eicosanoid production varies by sex in mesenteric ischemia reperfusion injury.

Intestinal ischemia/reperfusion (I/R)-induced injury is an inflammatory response with significant morbidity and mortality. The early inflammatory response includes neutrophil infiltration. However, the majority of rodent studies utilize male mice despite a sexual dimorphism in intestinal I/R-related diseases. We hypothesized that sex may alter inflammation by changing neutrophil infiltration and eicosanoid production. To test this hypothesis, male and female C57Bl/6 mice were subjected to sham treatment or 30 min intestinal ischemia followed by a time course of reperfusion. We demonstrate that compared to male mice, females sustain significantly less intestinal I/R-induced tissue damage and produced significant LTB4 concentrations. Male mice release PGE2. Finally, treatment with a COX-2 specific inhibitor, NS-398, attenuated I/R-induced injury, total peroxidase level, and PGE2 production in males, but not in similarly treated female mice. Thus, I/R-induced eicosanoid production and neutrophil infiltration varies between sexes suggesting that distinct therapeutic intervention may be needed in clinical ischemic diseases.

Cholesterol crystals use complement to increase NLRP3 signaling pathways in coronary and carotid atherosclerosis.

BACKGROUND: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease. METHODS: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling. FINDINGS: Transcripts of interleukin (IL)-1beta(ß) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1ß protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1ß, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components. INTERPRETATION: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

COVID-19 cytokine storm: The anger of inflammation.

Patients with COVID-19 who require ICU admission might have the cytokine storm. It is a state of out-of-control release of a variety of inflammatory cytokines. The molecular mechanism of the cytokine storm has not been explored extensively yet. The attachment of SARS-CoV-2 spike glycoprotein with angiotensin-converting enzyme 2 (ACE2), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. Four molecular axes that may be involved in SARS-CoV-2 driven inflammatory cytokine overproduction are addressed in this work. The virus-mediated down-regulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ACE/angiotensin II/AT1R axis), attenuation of Mas receptor (ACE2/MasR axis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis), and activation of the complement system including C5a and C5b-9 components. The molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat COVID-19 associated acute respiratory distress syndrome.

In response to complement anaphylatoxin peptides C3a and C5a, human vascular endothelial cells migrate and mediate the activation of B-cells and polarization of T-cells.

The vascular endothelium has been discovered in the past several years to be important in shaping the cellular immune response. During the immune response the vascular endothelium is constantly perturbed by biologically potent molecules, including the complement activation peptides, C3a and C5a. Despite the importance of C3a and C5a in inflammation and immunity, their role in modulating lymphocyte function via activation of vascular endothelial cells is unknown. Accordingly, we investigated the regulated expression of the C3a and C5a receptors (complement anaphylatoxin C3a receptor [C3aR] and complement anaphylatoxin C5a receptor 1 [C5aR1]) on human umbilical vascular endothelial cells (HUVECs) and examined how C3a or C5a activation of HUVECs affects the activation and polarization of lymphatic cells. Our findings demonstrated that C3a and C5a increase C3aR and C5aR1 expression by HUVECs as well as directing their cellular transmigration and spreading through transwell filters. Moreover, C3a- or C5a-stimulated endothelial cells: (1) caused activation of B-lymphoblasts with significant increase in Fas Ligand (CD95L) (FasL), CD69, and IL-R1 expression, and (2) skewed T-lymphoblast cells toward a Th1 subtype, (CD4+ /CCR5+ ) that correlated with significant increase of IFN-γ. Collectively, these data indicate that C3a and C5a signaling is important in the activation and polarization of lymphocytes as they traffic through the vascular endothelium during the immune response.

[Effect of complement C5a on the expression of MCP-1 and NGAL in immune kidney injury of trichloroethylene sensitized mice].

Objective: To explore the possible role of C5a in the pathogenesis of renal injury in TCE- sensitized mice, to analyze the impact of expression of neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemotactic protein-1 (MCP-1) in the presence or absence of C5a receptor antagonist (C5aRA) pretreatment. Methods: A total of 50 female specific pathogens free(SPF) BALB/c mice were randomly divided into blank control group (n=5) , solvent control group (n=5) , TCE group (n=20) , and TCE+C5aRA group (n= 20) . After one week for adaptive feeding, a mouse model of TCE-induced skin sensitization was established by treating with 50% TCE and 30% TCE in turn. The mice in solvent control group accept same reagents without TCE and the mice in blank control group underwent nothing. In TCE +C5aRA group, except for the TCE solution treatment, mice were intraperitoneally injected with 0.5 mg/kg C5aRA solution at the time of challenge. And the skin erythema and edema reaction were scored 24 h after the last challenge. The mice were divided into sensitization positive group and sensitization negative group according to the scoring result. The mice were aseptically sacrificed 72 h after the last challenge to obtain the kidneys. The structural damage of kidney was observed after histopathological staining. The levels of NGAL and MCP-1 mRNA and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) , respectively. Results: The sensitization rate of mice in TCE group and TCE+C5aRA group was 45.0% (9/20) and 40.0% (8/20) , respectively. No skin lesions was found in the mice of blank control group and solvent control group. The results of histopathological staining showed that the TCE sensitization positive mice showed renal tubular dilatation, vacuolar degeneration of renal tubular epithelial cells, and infiltration of interstitial cells. The pathological damage of the kidney in TCE sensitization positive group was mild, and no inflammatory cell infiltration was seen. The data of qRT-PCR showed that the expression levels of NGAL and MCP-1 mRNA in the TCE sensitization positive group were significantly increased than in solvent control group and TCE sensitization negative group (P<0.05) , while the levels of NGAL and MCP-1 mRNA in TCE+C5aRA sensitization positive group were decreased than TCE sensitization positive group (P <0.05) . The results of IHC showed that the expression levels of NGAL and MCP-1 in TCE protein sensitization positive group were significantly higher than those in solvent control group and TCE sensitization negative group (P<0.05) . After C5aRA pretreatment, the expression levels of NGAL and MCP-1 protein were decreased than the mice in TCE sensitization positive group (P<0.05) . Conclusion: The regulation of C5a on the expression of MCP-1 and NGAL may participate in TCE- induced mice kidney damage, and pharmacological inhibition of C5a seems to be an effective way to protect the kidney injury in TCE-sensitized mice.

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.

PODXL1 promotes metastasis of the pancreatic ductal adenocarcinoma by activating the C5aR/C5a axis from the tumor microenvironment.

Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors.

C5a/C5aR1 Pathway Is Critical for the Pathogenesis of Psoriasis.

Psoriasis is one of the most common chronic inflammatory skin diseases, affecting ~2% of the population. The lack of characterization of the pathogenesis of psoriasis has hindered efficient clinical treatment of the disease. In our study, we observed that expression of complement component 5a receptor 1(C5aR1) was significantly increased in skin lesions of both imiquimod (IMQ) and IL23-induced psoriatic mice and patients with psoriasis. C5aR1 deficiency or treatment with C5a receptor 1 antagonist (C5aR1a) in mice significantly attenuated psoriasis-like skin lesions and expression of inflammatory cytokines and chemokines. Moreover, C5aR1 deficiency significantly decreased IMQ-induced infiltration of plasmacytoid dendritic cells (pDCs), monocytes and neutrophils in psoriatic skin lesions and functions of pDCs, evidenced by the remarkable reduction in the IMQ-induced production of interferon-α (IFN-α) and tumor necrosis factor α (TNF-α), and FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent pDCs differentiation. Accordingly, in vitro treatment with recombinant C5a accelerated pDCs migration and the differentiation of bone marrow cells into pDCs. Furthermore, biopsies of psoriatic patients showed a dramatic increase of C5aR1+ pDCs infiltration in psoriatic skin lesions, compared to healthy subjects. Our results provide direct evidence that C5a/C5aR1 signaling plays a critical role in the pathogenesis of psoriasis. Inhibition of C5a/C5aR1 pathway is expected to be beneficial in the treatment of patients with psoriasis.

C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

The canonical complement component 5a (C5a) receptor (C5aR) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C5aR2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both C5aR1 and C5aR2 and signal through ERK and p38 MAPKs in response to C5a. Despite this, C5a had no impact on melanoma cell proliferation or migration in vitro. In vivo studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C5aR2-/- mice but reduced in C5aR1-/- mice and wild-type mice treated with a C5aR1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C5aR2-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C5aR1-/- mice, whereas total (CD3+) T lymphocytes and CD4+ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C5aR1. The results suggest that C5aR1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C5aR2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C5aR1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

Neutrophils from ANCA-associated vasculitis patients show an increased capacity to activate the complement system via the alternative pathway after ANCA stimulation.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), are autoimmune conditions associated with small vessel inflammation. Earlier studies indicate that complement activation via the alternative pathway plays a major role in the pathogenesis. In this study we have investigated if ANCA-activation of neutrophils from AAV patients leads to activation of the alternative complement pathway. C5a-primed neutrophils (PMN) from 10 AAV patients and 10 healthy controls (HC) were stimulated with PMA or IgG purified from PR3-ANCA positive patients (ANCA IgG). The supernatants were analyzed for release of complement proteins and markers of different granules by ELISA, and release of microparticles (MP) by flow cytometry. The ability of the supernatants to activate the alternative complement pathway was determined by incubation with normal serum and C3bBbP and C5a were measured by ELISA. MP were analyzed by flow cytometry and removed by centrifugation. The supernatants from the AAV patients' neutrophils produced significantly more C3bBbP compared with HCs (p = 0.0001). C3bBbP levels correlated with the number of MP. After removal of MP from the supernatants, alternative pathway activation was significantly lower. This study shows that primed and ANCA-stimulated neutrophils from AAV patients have a greater ability to activate the alternative complement pathway compared to primed neutrophils from healthy controls. This finding emphasizes the role of complement in the pathogenesis of AAV - underlining the therapeutic potential of C5a and other complement blockade.

Targeting the Complement Pathway as a Therapeutic Strategy in Lung Cancer.

Lung cancer is the leading cause of cancer death in men and women. Lung adenocarcinoma (LUAD), represents approximately 40% of all lung cancer cases. Advances in recent years, such as the identification of oncogenes and the use of immunotherapies, have changed the treatment of LUAD. Yet survival rates still remain low. Additionally, there is still a gap in understanding the molecular and cellular interactions between cancer cells and the immune tumor microenvironment (TME). Defining how cancer cells with distinct oncogenic drivers interact with the TME and new strategies for enhancing anti-tumor immunity are greatly needed. The complement cascade, a central part of the innate immune system, plays an important role in regulation of adaptive immunity. Initially it was proposed that complement activation on the surface of cancer cells would inhibit cancer progression via membrane attack complex (MAC)-dependent killing. However, data from several groups have shown that complement activation promotes cancer progression, probably through the actions of anaphylatoxins (C3a and C5a) on the TME and engagement of immunoevasive pathways. While originally shown to be produced in the liver, recent studies show localized complement production in numerous cell types including immune cells and tumor cells. These results suggest that complement inhibitory drugs may represent a powerful new approach for treatment of NSCLC, and numerous new anti-complement drugs are in clinical development. However, the mechanisms by which complement is activated and affects tumor progression are not well understood. Furthermore, the role of local complement production vs. systemic activation has not been carefully examined. This review will focus on our current understanding of complement action in LUAD, and describe gaps in our knowledge critical for advancing complement therapy into the clinic.

Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a--C5aR1 Pathway.

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow-derived macrophages from BVR fl/fl and LysM-Cre:BVR fl / fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVR fl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVR fl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a-C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization-associated chemokines, the levels of which depend in part on C5aR1 signaling.

Association of age-related macular degeneration with complement activation products, smoking, and single nucleotide polymorphisms in South Carolinians of European and African descent.

Purpose: Smoking and the incidence of age-related macular degeneration (AMD) have been linked to an overactive complement system. Here, we examined in a retrospective cohort study whether AMD-associated single nucleotide polymorphisms (SNPs), smoking, ethnicity, and disease status are correlated with blood complement levels. Methods: Population: The study involved 91 AMD patients and 133 controls, which included 73% Americans of European descent (EUR) and 27% Americans of African descent (AFR) in South Carolina. Readouts: Participants were genotyped for 10 SNPs and systemic levels of complement factor H (CFH) activity, and the complement activation products C3a, C5a, and Bb were assessed. Main Outcome Measures: Univariate and multivariable logistic regression models were used to examine associations between AMD status and distinct readouts. Results: AMD affects EUR individuals more than AFRs. EUR but not AFR AMD subjects revealed higher levels of Factors C3a and Bb. In all subjects, a 10-unit increase in C3a levels was associated with an approximately 10% increase in the odds of being AMD-positive, and C3a and Bb were associated with smoking. While CFH activity levels were not correlated with AMD, a significant interaction was evident between patient age and CFH activity. Finally, EURs had lower odds of AMD with enhanced copies of rs1536304 (VEGFA) and higher odds with more copy numbers of rs3766404 (CFH). Conclusions: Our results support previous studies of systemic complement components being potential biomarkers for AMD, but they suggest that smoking and disease do not synergistically affect complement levels. We also suggest a novel susceptibility and protective haplotypes in the South Carolinian AMD population. Our studies indicate that augmented complement activation associated with advanced AMD could be attributed to a decrease in CFH activity in younger patients.

The relationship of anti-phospholipase A2 receptor antibody and C5a complement with disease activity and short-term outcome in idiopathic membranous nephropathy.

BACKGROUND: The binding of anti-phospholipase A2 receptor (anti-PLA2R) antibody to podocyte and complement activation is the mechanisms of idiopathic membranous nephropathy (IMN). C5a, a complement activation end product, is a strong inflammatory cell stimulator and can influence the behavior of T cells and dendritic cells. This study examined the etiology-disease relationship and significance of auto-antibody and C5a with short-term remission. METHOD: Plasma anti-PLA2R antibody and C5a were measured with the blood samples that were collected when patients were admitted for renal biopsy. The deposition of IgA, IgG, IgM, C1q and C3c in glomerulus was graded according to immunofluorescence staining. The relationship of anti-PLA2R antibody with C5a, glomerular immunoglobulin and complement deposition was examined. Antibody and C5a levels as predictors of short-term remission were also examined. RESULTS: In 72 IMN patients, 50 patients had positive plasma anti-PLA2R antibody. The antibody had positive correlation to proteinuria. Patients with high grade IgG or C3c, but not IgA/IgM/C1q, deposition had higher anti-PLA2R antibody titers. C5a was increased in IMN patients, but had no correlation with anti-PLA2R antibody or proteinuria. The analysis revealed that C5a, not initial anti-PLA2R antibody, was a predictor associated with 12-month remission in patients receiving immunosuppression with multivariate-adjusted OR 0.74 (95% CI, 0.58-0.94, P = 0.01). CONCLUSION: This study provides indirect evidences of etiology-disease relationship of anti-PLA2R antibody in IMN patients. The role of C5a, a predictor of remission, in the disease course of MN and the influences on inflammatory cells in MN patients is worth to be clarified.