Macrophages promote heat stress nephropathy in mice via the C3a-C3aR-TNF pathway.

Heat-stress nephropathy (HSN) is associated with recurrent dehydration. However, the mechanisms underlying HSN remain largely unknown. In this study, we evaluated the role of dehydration in HSN and kidney injury in mice. Firstly, we found that complement was strongly activated in the mice that were exposed to dehydration; and among complement components, the interaction between C3a and its receptor, C3aR, was more closely associated with kidney injury. Then two-month-old mice were intraperitoneally injected with 2% dimethyl sulfoxide (DMSO) or the C3aR inhibitor SB290157 during dehydration. DMSO-treated mice exhibited excessive macrophage infiltration, renal cell apoptosis, and kidney fibrosis. In contrast, SB290157-treated mice had no apparent kidney injury. By fluorescence-activated cell sorting (FACS), we found that SB290157 treatment in mice remarkably inhibited macrophage infiltration and suppressed CCR2 expression in macrophages. In addition, C3a binding to C3aR promoted macrophage polarization toward the M1 phenotype and increased the production of TNF-α, which induced renal tubular epithelial cell (RTEC) apoptosis in vivo and in vitro. Interestingly, C3a treatment failed to directly induce TNF-α production and apoptosis in RTECs. However, TNF-α production in response to C3a treatment was significantly elevated when RTECs were cocultured with macrophages, suggesting that macrophages rather than RTECs are the target of C3a-C3aR interaction. At last, we proved that infusion of macrophages which highly expressed TNF-α would significantly deteriorate HSN in TNF-KO mice when they were exposed to recurrent dehydration. This study uncovers a novel mechanism underlying the pathogenesis of HSN, and a potential pathway to prevent kidney injury during dehydration.

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent.

Astrocytes perform a range of homeostatic and regulatory tasks that are critical for normal functioning of the central nervous system. In response to an injury or disease, astrocytes undergo a pronounced transformation into a reactive state that involves changes in the expression of many genes and dramatically changes astrocyte morphology and functions. This astrocyte reactivity is highly dependent on the initiating insult and pathological context. C3a is a peptide generated by the proteolytic cleavage of the third complement component. C3a has been shown to exert neuroprotective effects, stimulate neural plasticity and promote astrocyte survival but can also contribute to synapse loss, Alzheimer's disease type neurodegeneration and blood-brain barrier dysfunction. To test the hypothesis that C3a elicits differential effects on astrocytes depending on their reactivity state, we measured the expression of Gfap, Nes, C3ar1, C3, Ngf, Tnf and Il1b in primary mouse cortical astrocytes after chemical ischemia, after exposure to lipopolysaccharide (LPS) as well as in control naïve astrocytes. We found that C3a down-regulated the expression of Gfap, C3 and Nes in astrocytes after ischemia. Further, C3a increased the expression of Tnf and Il1b in naive astrocytes and the expression of Nes in astrocytes exposed to LPS but did not affect the expression of C3ar1 or Ngf. Jointly, these results provide the first evidence that the complement peptide C3a modulates the responses of astrocytes in a highly context-dependent manner.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

The role and mechanisms of Microglia in Neuromyelitis Optica Spectrum Disorders.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neurological disease that can cause blindness and disability. As the major mediators in the central nervous system, microglia plays key roles in immunological regulation in neuroinflammatory diseases, including NMOSD. Microglia can be activated by interleukin (IL)-6 and type I interferons (IFN-Is) during NMOSD, leading to signal transducer and activator of transcription (STAT) activation. Moreover, complement C3a secreted from activated astrocytes may induce the secretion of complement C1q, inflammatory cytokines and progranulin (PGRN) by microglia, facilitating injury to microglia, neurons, astrocytes and oligodendrocytes in an autocrine or paracrine manner. These processes involving activated microglia ultimately promote the pathological course of NMOSD. In this review, recent research progress on the roles of microglia in NMOSD pathogenesis is summarized, and the mechanisms of microglial activation and microglial-mediated inflammation, and the potential research prospects associated with microglial activation are also discussed.

Dynamic regulation of B cell complement signaling is integral to germinal center responses.

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.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

C3a and C5a facilitates the metastasis of myeloma cells by activating Nrf2.

Multiple myeloma (MM) is still an incurable hematological malignancy, with even poorer prognosis in MM patients with distant invasion. The present study was designed to explore the effects of C3a and C5a on the migration, invasion, and adhesion of MM tumor cells and to investigate the underlying mechanisms. As a result, the levels of C3a and C5a in plasma of MM patients were significantly higher than those of healthy donors. Consistently, the expression of C3a and C5a receptors on myeloma cells of MM patients was also significantly higher than that on sorted plasma cells of normal donors. C3a and C5a have been confirmed to increase the migration, invasion and adhesion of MM cell lines by activating the MEK/ERK pathway and increasing the nuclear transfer of Nrf2 in vitro. Moreover, the MM cell line U266 with Nrf2 downregulation was incubated with C3a and C5a, followed by injection into the tail vein of NOD-SCID mice. We found that Nrf2 downregulation attenuated the migration of anaphylatoxin C3a and C5a to MM tumor cells in bone marrow, liver and lung in vivo. In conclusion, our results indicate that activation of the complement cascade in MM patients may contribute to the migration, invasion and adhesion of MM cells, and this type of tumor cells dissemination in MM is, at least partially, regulated by Nrf2. Thereby, complement suppression or Nrf2 downregulation might offer a novel therapeutic opportunity for MM.

COVID-19: A collision of complement, coagulation and inflammatory pathways.

COVID-19 is frequently accompanied by a hypercoagulable inflammatory state with microangiopathic pulmonary changes that can precede the diffuse alveolar damage characteristic of typical acute respiratory distress syndrome (ARDS) seen in other severe pathogenic infections. Parallels with systemic inflammatory disorders such as atypical hemolytic uremic syndrome (aHUS) have implicated the complement pathway in the pathogenesis of COVID-19, and particularly the anaphylatoxins C3a and C5a released from cleavage of C3 and C5, respectively. C5a is a potent cell signalling protein that activates a cytokine storm-a hyper-inflammatory phenomenon-within hours of infection and the innate immune response. However, excess C5a can result in a pro-inflammatory environment orchestrated through a plethora of mechanisms that propagate lung injury, lymphocyte exhaustion, and an immune paresis. Furthermore, disruption of the homeostatic interactions between complement and extrinsic and intrinsic coagulation pathways contributes to a net pro-coagulant state in the microvasculature of critical organs. Fatal COVID-19 has been associated with a systemic inflammatory response accompanied by a pro-coagulant state and organ damage, particularly microvascular thrombi in the lungs and kidneys. Pathologic studies report strong evidence of complement activation. C5 blockade reduces inflammatory cytokines and their manifestations in animal studies, and has shown benefits in patients with aHUS, prompting investigation of this approach in the treatment of COVID-19. This review describes the role of the complement pathway and particularly C5a and its aberrations in highly pathogenic virus infections, and therefore its potential as a therapeutic target in COVID-19.

The Potential Role of Complement System in the Progression of Ovarian Clear Cell Carcinoma Inferred from the Gene Ontology-Based Immunofunctionome Analysis.

Ovarian clear cell carcinoma (OCCC) is the second most common epithelial ovarian carcinoma (EOC). It is refractory to chemotherapy with a worse prognosis after the preliminary optimal debulking operation, such that the treatment of OCCC remains a challenge. OCCC is believed to evolve from endometriosis, a chronic immune/inflammation-related disease, so that immunotherapy may be a potential alternative treatment. Here, gene set-based analysis was used to investigate the immunofunctionomes of OCCC in early and advanced stages. Quantified biological functions defined by 5917 Gene Ontology (GO) terms downloaded from the Gene Expression Omnibus (GEO) database were used. DNA microarray gene expression profiles were used to convert 85 OCCCs and 136 normal controls into to the functionome. Relevant offspring were as extracted and the immunofunctionomes were rebuilt at different stages by machine learning. Several dysregulated pathogenic functions were found to coexist in the immunopathogenesis of early and advanced OCCC, wherein the complement-activation-alternative-pathway may be the headmost dysfunctional immunological pathway in duality for carcinogenesis at all OCCC stages. Several immunological genes involved in the complement system had dual influences on patients' survival, and immunohistochemistrical analysis implied the higher expression of C3a receptor (C3aR) and C5a receptor (C5aR) levels in OCCC than in controls.

C3a receptor blockade protects podocytes from injury in diabetic nephropathy.

Renal activation of the complement system has been described in patients with diabetic nephropathy (DN), although its pathological relevance is still ill-defined. Here, we studied whether glomerular C3a, generated by uncontrolled complement activation, promotes podocyte damage, leading to proteinuria and renal injury in mice with type 2 diabetes. BTBR ob/ob mice exhibited podocyte loss, albuminuria, and glomerular injury accompanied by C3 deposits and increased C3a and C3a receptor (C3aR) levels. Decreased glomerular nephrin and α-actinin4 expression, coupled with integrin-linked kinase induction, were also observed. Treatment of DN mice with a C3aR antagonist enhanced podocyte density and preserved their phenotype, limiting proteinuria and glomerular injury. Mechanistically, ultrastructural and functional mitochondrial alterations, accompanied by downregulation of antioxidant superoxide dismutase 2 (SOD2) and increased protein oxidation, occurred in podocytes and were normalized by C3aR blockade. In cultured podocytes, C3a induced cAMP-dependent mitochondrial fragmentation. Alterations of mitochondrial membrane potential, SOD2 expression, and energetic metabolism were also found in response to C3a. Notably, C3a-induced podocyte motility was inhibited by SS-31, a peptide with mitochondrial protective effects. These data indicate that C3a blockade represents a potentially novel therapeutic strategy in DN for preserving podocyte integrity through the maintenance of mitochondrial functions.

Integrating complement into the molecular pathogenesis of Hidradenitis Suppurativa.

Complement inhibition has been identified as a potential therapeutic target for multiple inflammatory disorders including Hidradenitis Suppurativa (HS). It is currently unclear how complement integrates into our current model of molecular pathogenesis in HS and whether it represents a central component of pathogenesis, or a neutrophil-associated bystander. Levels of C5a in serum and tissue correlate with disease activity and degree of neutrophilic infiltrates in HS. C5a has been associated with Th17 immune axis activation in psoriasis, rheumatoid arthritis and Crohn's disease with strong similarities to TH17 activation in HS. Porphyromonas species (which are identified in the HS microbiome) are able to cleave inactive C5 into C5a implicating the cutaneous microbiome as an activator of complement. C3a and C5a are associated with activation of the NLRP3 inflammasome, implicated in the inflammatory drive in HS. Complement receptors are present upon dendritic cells, monocytes, fibroblasts and adipocytes, which may broaden the potential contribution of complement to multiple aspects of HS pathogenesis. Dysregulation of complement receptor pathways has been documented in obesity, insulin resistance and polycystic ovarian syndrome leading to the possibility that complement may explain the epidemiological associations between these conditions and HS. The therapeutic potential of complement inhibitors in HS may be related to the therapeutic target (complement receptor or complement subunit) and the presence of alternate receptors (such as C5aR2) or ligands (including C3a, PAMPs and DAMPs). Integrating complement into the known pathogenesis of HS may aid in explaining the contradictory results between Phase 2 studies of C5a antagonists. It also allows for the identification of existing knowledge gaps to target further clinical investigation and research.

Immune and Inflammatory Proteins in Cord Blood as Predictive Biomarkers of Retinopathy of Prematurity in Preterm Infants.

Purpose: To determine whether elevated levels of immune/inflammatory proteins in cord blood, alone or in combination with conventional clinical parameters, can predict the occurrence and progression of retinopathy of prematurity (ROP) in preterm infants. Methods: This was a retrospective cohort study of 110 premature singleton infants who were born at ≤32.0 weeks. Cord plasma at birth was assayed for interleukin-6, C3a, C5a, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1, macrophage colony-stimulating factor, endostatin, a proliferation-inducing ligand, insulin-like growth factor-binding protein-1 (IGFBP-1), IGFBP-2, and calcium-binding protein A8/A9 complex levels. The primary outcome measures were the occurrence of any stage ROP, severe ROP (>stage 3), and vision-threatening type 1 ROP requiring laser treatment. Results: ROP was diagnosed in 30 of 110 infants (27.3%), including 14 (12.7%) with severe ROP. Laser treatment was performed on 7 infants (6.4%). Multiple logistic regression analyses indicated that elevated levels of cord plasma IL-6 were significantly associated with severe ROP, whereas elevated levels of cord plasma C5a were significantly associated with ROP laser treatments. However, none of the proteins measured in the cord plasma were associated with ROP occurrence. Using a stepwise regression procedure, we developed a combined prediction model, which included high cord plasma IL-6 levels and low birth weight for severe ROP (area under the curve [AUC], 0.840), and high cord plasma C5a levels and low birth weight for laser treatment (AUC, 0.884). Conclusions: Elevated levels of cord plasma IL-6 and C5a could be used as independent markers to predict severe ROP and laser treatment, respectively, with combined models predicting ROP progression with good accuracy.

Complement activation sustains neuroinflammation and deteriorates adult neurogenesis and spatial memory impairment in rat hippocampus following sleep deprivation.

BACKGROUND: An association between neuroinflammation, reduced adult neurogenesis, and cognitive impairment has been established in sleep deprivation (SD). Complement receptors are expressed on neuronal and glial cells, thus, regulate the neuroinflammation, neurogenesis and learning/memory. However, understanding of the effect of SD on the brain-immune system interaction associated with cognitive dysfunction and its mechanisms is obscure. We hypothesized that complement activation induced changes in inflammatory and neurogenesis related proteins might be involved in the cognitive impairment during SD. METHODOLOGY: Adult male Sprague Dawley rats were used. Rats were sleep deprived for 48 h using a novel automated SD apparatus. Dosage of BrdU (50 mg/kg/day, i.p. in 0.07 N NaOH), complement C3a receptor antagonist (C3aRA; SB290157; 1 mg/kg/day, i.p.) in 1.16% v/v PBS and complement C5a receptor antagonist (C5aRA; W-54011; 1 mg/kg/day, i.p.) in normal saline were used. Rats were subjected to spatial memory evaluation following SD. Hippocampal tissue was collected for biochemical, molecular, and immunohistochemical studies. T-test and ANOVA were used for the statistical analysis. RESULTS: An up-regulation in the levels of complement components (C3, C5, C3a, C5a) and receptors (C3aR and C5aR) in hippocampus, displayed the complement activation during SD. Selective antagonism of C3aR/C5aR improved the spatial memory performance of sleep-deprived rats. C3aR antagonist (C3aRA) or C5aR antagonist (C5aRA) treatment inhibited the gliosis, maintained inflammatory cytokines balance in hippocampus during SD. Complement C3aR/C5aR antagonism improved hippocampal adult neurogenesis via up-regulating the BDNF level following SD. Administration of C3aRA and C5aRA significantly maintained synaptic homeostasis in hippocampus after SD. Gene expression analysis showed down-regulation in the mRNA levels of signal transduction pathways (Notch and Wnt), differentiation and axogenous proteins, which were found to be improved after C3aRA/C5aRA treatment. These findings were validated at protein and cellular level. Changes in the corticosterone level and ATP-adenosine-NO pathway were established as the key mechanisms underlying complement activation mediated consequences of SD. CONCLUSION: Our study suggests complement (C3a-C3aR and C5a-C5aR) activation as the novel mechanism underlying spatial memory impairment via promoting neuroinflammation and adult neurogenesis decline in hippocampus during SD, thereby, complement (C3aR/C5aR) antagonist may serve as the novel therapeutics to improve the SD mediated consequences.

C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis.

Chronic tubulointerstitial injury impacts the prognosis of focal segmental glomerulosclerosis (FSGS). We found that the level of versican V1 was increased in tubular cells of FSGS patients. Tubular cell-derived versican V1 induced proliferation and collagen synthesis by activating the CD44/Smad3 pathway in fibroblasts. Both urine C3a and suPAR were increased and bound to the tubular cells in FSGS patients. C3a promoted the transcription of versican by activating the AKT/ß-catenin pathway. C3aR knockout decreased the expression of versican in Adriamycin-treated (ADR-treated) mice. On the other hand, suPAR bound to integrin ß6 and activated Rac1, which bound to SRp40 at the 5' end of exon 7 in versican pre-mRNA. This binding inhibited the 3'-end splicing of intron 6 and the base-pair interactions between intron 6 and intron 8, leading to the formation of versican V1. Cotreatment with ADR and suPAR specifically increased the level of versican V1 in tubulointerstitial tissues and caused more obvious interstitial fibrosis in mice than treatment with only ADR. Altogether, our results show that C3a and suPAR drive versican V1 expression in tubular cells by promoting transcription and splicing, respectively, and the increases in tubular cell-derived versican V1 induce interstitial fibrosis by activating fibroblasts in FSGS.

Complement factors C3a and C5a mimick a proinflammatory microenvironment and increase HBV IGRA sensitivity.

BACKGROUND: Hepatitis B virus (HBV) infections represent a global health problem and chronic hepatitis B (CHB) leads to liver cirrhosis and hepatocellular carcinoma. Thus, timely diagnosis of hepatitis B is crucial to ensure adequate treatment. We developed a powerful and rapid whole blood-based cytokine release assay assessing cellular immune responses to HBV antigens. IL-2 and IFNγ release in this assay depicts hepatitis B vaccination status. Of note, CHB goes along with elevated C5a concentrations in plasma. We aim at mimicking the proinflammatory microenvironment associated with HBV infection to enhance the diagnostic quality of our HBV specific cytokine release assay. We specifically investigated the potential of the complement factors C3a and C5a as costimulators and analyzed their potential effects on activation marker expression on T cells and antigen presenting cells. RESULTS: Whole blood from 87 healthy individuals (n = 59 hepatitis B vaccinated, n = 28 unvaccinated) was stimulated with HBV surface antigen (HBsAg) in presence or absence of C3a or C5a, respectively. Further, C3a and C5a were used in combination to investigate potential synergistic effects. IL-2 and IFNγ levels in plasma were quantified using ELISA. Complement factor C5a specifically enhances HBsAg-mediated IL-2 (690.3 ± 195.4 pg/ml vs. 789.4 ± 216.5 pg/ml) and IFNγ (146.0 ± 43.1 pg/ml vs. 336.7 ± 67.9 pg/ml) responses in whole blood. Similar cytokine levels were measured when both C3a and C5a were used. With a diagnostic specificity of 90% the IFNγ release assay reached a diagnostic sensitivity of 49.2% upon whole blood stimulation with HBsAg alone, but of 78.9% when HBsAg was combined with C3a and C5a. CONCLUSIONS: Innate signals mediated via complement pathways contribute to HBV-specific cellular immune responses. The massively improved diagnostic sensitivity of the IFNγ release assay after addition of C3a and C5a demonstrates that these effects render whole blood-based cytokine release assays even more potent as screening tools in HBV immunology and HBV vaccination studies.

Complement anaphylatoxins C3a and C5a: Emerging roles in cancer progression and treatment.

Recent insights into the role of complement anaphylatoxins C3a and C5a in cancer provide new opportunities for the development of innovative biomarkers and therapeutic strategies. These two complement activation products can maintain chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and increase the motility and metastatic potential of cancer cells. Still, the diverse heterogeneity of responses mediated by these peptides poses a challenge both to our understanding of the role played by these molecules in cancer progression and to the development of effective treatments. This review attempts to summarize the evidence surrounding the involvement of anaphylatoxins in the biological contexts associated with tumor progression. We also describe the recent developments that support the inhibition of anaphylatoxins, or their cognate receptors C3aR and C5aR1, as a treatment option for maximizing the clinical efficacy of current immunotherapies that target the PD-1/PD-L1 immune checkpoint.

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.

Inflammatory and Immune Proteins in Umbilical Cord Blood: Association with Hearing Screening Test Failure in Preterm Neonates.

OBJECTIVE: We aimed to determine whether elevated levels of various inflammatory and immune proteins in umbilical cord blood are associated with an increased risk of newborn hearing screening (NHS) test failure in preterm neonates. METHODS: This retrospective cohort study included 127 premature singleton infants who were born at ≤33.6 weeks. Umbilical cord plasma at birth was assayed for interleukin (IL)-6, complement C3a and C5a, matrix metalloproteinase (MMP)-9, macrophage colony-stimulating factor (M-CSF), and endostatin levels using ELISA kits. Neonatal blood C-reactive protein (CRP) levels were measured within 2 hours of birth. The primary outcome measure was a uni- or bilateral refer result on an NHS test. Univariate and multivariate analyses were applied. RESULTS: Fifteen (11.8%) infants failed the NHS test. In the univariate analyses, high IL-6 and low C3a levels in umbilical cord plasma, funisitis, and an elevated CRP level (>5 mg/L) in the immediate postnatal period were significantly associated with NHS test failure. However, the levels of umbilical cord plasma MMP-9, C5a, M-CSF, and endostatin were not significantly different between infants who passed and those who failed the NHS test. Multiple logistic regression analyses indicated that elevated umbilical cord plasma C3a levels were independently associated with a reduced risk of NHS test failure, whereas elevated levels of umbilical cord plasma IL-6 and high CRP levels in the immediate postnatal period were significantly associated with NHS test failure. CONCLUSIONS: Our data demonstrated that in preterm neonates, a systemic fetal inflammatory response reflected by umbilical cord plasma IL-6 and immediate postnatal CRP levels may contribute to the risk for NHS test failure, whereas the changes in complement activation fragments initiated in utero may have protective effect of hearing screen failure.

C3a is required for ILC2 function in allergic airway inflammation.

Aberrant type 2 responses underlie the pathologies in allergic diseases like asthma, yet, our understanding of the mechanisms that drive them remains limited. Recent evidence suggests that dysregulated innate immune factors can perpetuate asthma pathogenesis. In susceptible individuals, allergen exposure triggers the activation of complement, a major arm of innate immunity, leading to the aberrant generation of the C3a anaphylatoxin. C3 and C3a have been shown to be important for the development of Th2 responses, yet remarkably, the mechanisms by which C3a regulates type 2 immunity are relatively unknown. We demonstrate a central role for C3a in driving type 2 innate lymphoid cells (ILC2)-mediated inflammation in response to allergen and IL-33. Our data suggests that ILC2 recruitment is C3a-dependent. Further, we show that ILC2s directly respond to C3a, promoting type 2 responses by specifically: (1) inducing IL-13 and granulocyte-macrophage colony-stimulating factor, whereas inhibiting IL-10 production from ILC2; and (2) enhancing their antigen-presenting capability during ILC-T-cell cross-talk. In summary, we identify a novel mechanism by which C3a can mediate aberrant type 2 responses to aeroallergen exposure, which involves a yet unrecognized cross-talk between two major innate immune components-complement and group 2 innate lymphoid cells.