Complement decay-accelerating factor inhibits inflammation-induced myopia development.

Myopia is regarded as a worldwide epidemic ocular disease, has been proved related to inflammation. CD55, also known as decay-accelerating factor (DAF) can modulate the activation of complement through inhibiting the formation of complement 3 convertase and its dysregulation is involved in various inflammatory diseases. To investigate the association between CD55 and myopia, and to test whether CD55 can inhibit myopia development by suppressing inflammation in the eye, we use three different animal models including monocular form-deprivation myopia, myopia induced by TNF-α administration and allergic conjunctivitis animal model to reveal the CD55 in myopia development. The tears of thirty-eight participants with different spherical equivalents were collected and CD55 in the tears were also analyzed. Complement 3 and complement 5 levels increased while CD55 levels decreased in allergic conjunctivitis and myopic eyes. After anti-inflammatory drugs administration, CD55 expression was increased in monocular form-deprivation myopia model. We also found inflammatory cytokines TGF-ß, IL-6, TNF-α, and IL-1ß may enhance complement 3 and complement 5 activation while CD55 level was suppressed contrary. Moreover, lower CD55 levels were found in the tears of patients with myopia with decreased diopter values. Finally, CD55-Fc administration on the eyelids can inhibit the elongation of axial length and change of refractive error. CD55-Fc application also suppress myopia development subsequent to complement 3 and complement 5 reduction and can lower myopia-specific (MMP-2 and TGF-ß) cytokine expression in TNF-α induced myopia animal model. This suggests that CD55 can inhibit myopia development by suppression of complement activation and eventual down-regulation of inflammation.

CD14 facilitates perinatal human cytomegalovirus infection in biliary epithelial cells via CD55.

Background & Aims: A high human cytomegalovirus (HCMV) infection rate accompanied by an increased level of bile duct damage is observed in the perinatal period. The possible mechanism was investigated. Methods: A total of 1,120 HCMV-positive and 9,297 HCMV-negative children were recruited, and depending on age, their liver biochemistry profile was compared. Fetal and infant biliary epithelial cells (F-BECs and I-BECs, respectively) were infected with HCMV, and the differences in cells were revealed by proteomic analysis. Protein-protein interactions were examined by coimmunoprecipitation and mass spectrometry analyses. A murine cytomegalovirus (MCMV) infection model was established to assess treatment effects. Results: Perinatal HCMV infection significantly increased the level of bile duct damage. Neonatal BALB/c mice inoculated with MCMV showed obvious inflammation in the portal area with an abnormal bile duct structure. Proteomics analysis showed higher CD14 expression in F-BECs than in I-BECs. CD14 siRNA administration hindered HCMV infection, and CD14-knockout mice showed lower MCMV-induced bile duct damage. HCMV infection upregulated CD55 and poly ADP-ribose polymerase-1 (PARP-1) expression in F-BECs. Coimmunoprecipitation and mass spectrometry analyses revealed formation of the CD14-CD55 complex. siRNA-mediated inhibition of CD55 expression reduced sCD14-promoted HCMV replication in F-BECs. In MCMV-infected mice, anti-mouse CD14 antibody and PARP-1 inhibitor treatment diminished cell death, ameliorated bile duct damage, and reduced mortality. Conclusions: CD14 facilitates perinatal HCMV infection in BECs via CD55, and PARP-1-mediated cell death was detected in perinatal cytomegalovirus-infected BECs. These results provide new insight into the treatment of perinatal HCMV infection with bile duct damage. Impact and implications: Perinatal human cytomegalovirus (HCMV) infection is associated with bile duct damage, but the underlying mechanism is still unknown. We discovered that CD14 expression is increased in biliary epithelial cells during perinatal HCMV infection and facilitates viral entry through CD55. We also detected PARP-1-mediated cell death in perinatal HCMV-infected biliary epithelial cells. We showed that blocking CD14 or inhibiting PARP-1 reduced bile duct damage and mortality in a mouse model of murine cytomegalovirus infection. Our findings provide a new insight into therapeutic strategies for perinatal HCMV infection.

A 21-bp deletion in the complement regulator CD55 promotor region is associated with multifocal motor neuropathy and its disease course.

BACKGROUND AND AIMS: To further substantiate the role of antibody-mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor polymorphisms of genes encoding the membrane-bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course. METHODS: We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters. RESULTS: The genotype frequencies of rs28371582, a 21-bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 [1.27-4.58]), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council [MRC] points/year; 95% confidence interval [CI]: 0.040-0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre-diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083-1.80, p .032). INTERPRETATION: MMN susceptibility is associated with a 21-bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long-term disease outcome. Taken together, these results point out the relevance of the pre-C5 level of the complement cascade in the inflammatory processes underlying MMN.

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

INTRODUCTION: Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. METHODS: EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. RESULTS: A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). CONCLUSION: Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes.

Overexpression of the inducible heme oxygenase (HO-1) isoform in visceral renal glomerular epithelial cells (podocytes) using in vivo transgenesis methods was shown to increase glomerular expression of the complement regulatory protein decay-accelerating factor (DAF, CD55) and reduce complement activation/deposition in a rat model of immune-mediated injury. In this preliminary study, we assessed whether constitutively expressed HO-1 regulates CD55 expression in cultured rat podocytes. We employed methods of flow cytometry, quantitative (q) RT-qPCR and post-transcriptional HO-1 gene silencing (HO-1 interfering RNA, RNAi), to assess changes in constitutive (basal) levels of podocyte HO-1 and CD55 mRNA in cultured rat podocytes. Additionally, the effect of the HO-1 inducer, heme, on HO-1 and CD55 expression was assessed. Results indicate that rat podocytes constitutively express HO-1 and DAF and that the HO-1 inducer, heme, increases both HO-1 and DAF expression. HO-1 gene silencing using RNA interference (RNAi) is feasible but the effect on constitutive CD55 transcription is inconsistent. These observations are relevant to conditions of podocyte exposure to heme that can activate the complementary cascade, as may occur in systemic or intraglomerular hemolysis.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

Metalloporphyrins Reduce Proteinuria in Podocyte Immune Injury: The Role of Metal and Porphyrin Moieties.

Depending on their central metal atom, metalloporphyrins (MPs) can attenuate or exacerbate the severity of immune-mediated kidney injury, and this has been attributed to the induction or inhibition of heme oxygenase (HO) activity, particularly the inducible isoform (HO-1) of this enzyme. The role of central metal or porphyrin moieties in determining the efficacy of MPs to attenuate injury, as well as mechanisms underlying this effect, have not been assessed. Using an antibody-mediated complement-dependent model of injury directed against rat visceral glomerular epithelial cells (podocytes) and two MPs (FePPIX, CoPPIX) that induce both HO-1 expression and HO enzymatic activity in vivo but differ in their chelated metal, we assessed their efficacy in reducing albuminuria. Podocyte injury was induced using rabbit immune serum raised against the rat podocyte antigen, Fx1A, and containing an anti-Fx1A antibody that activates complement at sites of binding. FePPIX or CoPPIX were injected intraperitoneally (5 mg/kg) 24 h before administration of the anti-Fx1A serum and on days 1, 3, 6, and 10 thereafter. Upon completion of urine collection on day 14, the kidney cortex was obtained for histopathology and isolation of glomeruli, from which total protein extracts were obtained. Target proteins were analyzed by capillary-based separation and immunodetection (Western blot analysis). Both MPs had comparable efficacy in reducing albuminuria in males, but the efficacy of CoPPIX was superior in female rats. The metal-free protoporphyrin, PPIX, had minimal or no effect on urine albumin excretion. CoPPIX was also the most potent MP in inducing glomerular HO-1, reducing complement deposition, and preserving the expression of the complement regulatory protein (CRP) CD55 but not that of CD59, the expression of which was reduced by both MPs. These observations demonstrate that the metal moiety of HO-1-inducing MPs plays an important role in reducing proteinuria via mechanisms involving reduced complement deposition and independently of an effect on CRPs.

Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells.

OBJECTIVES: This study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. STUDY DESIGN: We analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. RESULTS: We observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 & CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. CONCLUSION: The study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.

Adipose tissue-derived human mesenchymal stromal cells can better suppress complement lysis, engraft and inhibit acute graft-versus-host disease in mice.

BACKGROUND: Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transplantation of immunosuppressive human mesenchymal stromal cells (hMSCs) can protect against aGvHD post-HSCT; however, their efficacy is limited by poor engraftment and survival. Moreover, infused MSCs can be damaged by activated complement, yet strategies to minimise complement injury of hMSCs and improve their survival are limited. METHODS: Human MSCs were derived from bone marrow (BM), adipose tissue (AT) and umbilical cord (UC). In vitro immunomodulatory potential was determined by co-culture experiments between hMSCs and immune cells implicated in aGvHD disease progression. BM-, AT- and UC-hMSCs were tested for their abilities to protect aGvHD in a mouse model of this disease. Survival and clinical symptoms were monitored, and target tissues of aGvHD were examined by histopathology and qPCR. Transplanted cell survival was evaluated by cell tracing and by qPCR. The transcriptome of BM-, AT- and UC-hMSCs was profiled by RNA-sequencing. Focused experiments were performed to compare the expression of complement inhibitors and the abilities of hMSCs to resist complement lysis. RESULTS: Human MSCs derived from three tissues divergently protected against aGvHD in vivo. AT-hMSCs preferentially suppressed complement in vitro and in vivo, resisted complement lysis and survived better after transplantation when compared to BM- and UC-hMSCs. AT-hMSCs also prolonged survival and improved the symptoms and pathological features of aGvHD. We found that complement-decay accelerating factor (CD55), an inhibitor of complement, is elevated in AT-hMSCs and contributed to reduced complement activation. We further report that atorvastatin and erlotinib could upregulate CD55 and suppress complement in all three types of hMSCs. CONCLUSION: CD55, by suppressing complement, contributes to the improved protection of AT-hMSCs against aGvHD. The use of AT-hMSCs or the upregulation of CD55 by small molecules thus represents promising new strategies to promote hMSC survival to improve the efficacy of transplantation therapy. As complement injury is a barrier to all types of hMSC therapy, our findings are of broad significance to enhance the use of hMSCs for the treatment of a wide range of disorders.

Therapeutic hyperthermia regulates complement C3 activation and suppresses tumor development through HSPA5/NFκB/CD55 pathway in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is endemic in Southern China and Southeast Asia. Hyperthermia is widely used in combination with chemotherapy and radiotherapy to enhance therapeutic efficacy in NPC treatment, but the underlying anti-tumor mechanisms of hyperthermia remain unclear. Complement C3 has been reported to participate in the activation of immune system in the tumor microenvironment, leading to tumor growth inhibition. In this study, we aimed to explore the effect and mechanisms of hyperthermia and investigate the functional role of complement C3 in NPC hyperthermia therapy (HT). The serum levels of complement C3 before and after hyperthermia therapy in patients with NPC were analyzed. NPC cell lines SUNE1 and HONE1 were used for in vitro experiment to evaluate the function of complement C3 and HT on cell proliferation and apoptosis. SUNE1 xenograft mouse model was established and tumor-bearing mice were treated in water bath at a constant temperature of 43°C. Tumor samples were collected at different time points to verify the expression of complement C3 by immunohistochemical staining and western blot. The differential expressed genes after hyperthermia were analyzed by using RNA sequencing. We found that complement could enhance hyperthermia effect on suppressing proliferation and promoting apoptosis of tumor cells in NPC. Hyperthermia decreased the mRNA expression of complement C3 in tumor cells, but promoted the aggregation and activation circulating C3 in NPC tumor tissue. By using in vitro hyperthermia-treated NPC cell lines and SUNE1 xenograft tumor-bearing mice, we found that the expression of heat shock protein 5 (HSPA5) was significantly upregulated. Knockdown of HSPA5 abrogated the anti-tumor effect of hyperthermia. Moreover, we demonstrated that hyperthermia downregulated CD55 expression via HSPA5/NFκB (P65) signaling and activated complement cascade. Our findings suggest that therapeutic hyperthermia regulates complement C3 activation and suppresses tumor development via HSPA5/NFκB/CD55 pathway in NPC.

Excluding Digenic Inheritance of PGAP2 and PGAP3 Variants in Mabry Syndrome (OMIM 239300) Patient: Phenotypic Spectrum Associated with PGAP2 Gene Variants in Hyperphosphatasia with Mental Retardation Syndrome-3 (HPMRS3).

We present a case report of a child with features of hyperphosphatasia with neurologic deficit (HPMRS) or Mabry syndrome (MIM 239300) with variants of unknown significance in two post-GPI attachments to proteins genes, PGAP2 and PGAP3, that underlie HPMRS 3 and 4. BACKGROUND: In addition to HPMRS 3 and 4, disruption of four phosphatidylinositol glycan (PIG) biosynthesis genes, PIGV, PIGO, PIGW and PIGY, result in HPMRS 1, 2, 5 and 6, respectively. METHODS: Targeted exome panel sequencing identified homozygous variants of unknown significance (VUS) in PGAP2 c:284A>G and PGAP3 c:259G>A. To assay the pathogenicity of these variants, we conducted a rescue assay in PGAP2 and PGAP3 deficient CHO cell lines. RESULTS: Using a strong (pME) promoter, the PGAP2 variant did not rescue activity in CHO cells and the protein was not detected. Flow cytometric analysis showed that CD59 and CD55 expression on the PGAP2 deficient cell line was not restored by variant PGAP2. By contrast, activity of the PGAP3 variant was similar to wild-type. CONCLUSIONS: For this patient with Mabry syndrome, the phenotype is likely to be predominantly HPMRS3: resulting from autosomal recessive inheritance of NM_001256240.2 PGAP2 c:284A>G, p.Tyr95Cys. We discuss strategies for establishing evidence for putative digenic inheritance in GPI deficiency disorders.

miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

KLF4 and CD55 expression and function depend on each other.

The transcription factor Kruppel-like factor 4 (KLF4) regulates the expression of immunosuppressive and anti-thrombotic proteins. Despite its importance in maintaining homeostasis, the signals that control its expression and the mechanism of its transactivation remain unclarified. CD55 [aka decay accelerating factor (DAF)], now known to be a regulator of T and B cell responses, biases between pro- and anti-inflammatory processes by controlling autocrine C3a and C5a receptor (C3ar1/C5ar1) signaling in cells. The similarity in CD55's and KLF4's regulatory effects prompted analyses of their functional relationship. In vascular endothelial cells (ECs), CD55 upregulation accompanied KLF4 expression via a p-CREB and CREB Binding Protein (CBP) mechanism. In both ECs and macrophages, CD55 expression was essential for KLF4's downregulation of pro-inflammatory/pro-coagulant proteins and upregulation of homeostatic proteins. Mechanistic studies showed that upregulation of KLF4 upregulated CD55. The upregulated CD55 in turn enabled the recruitment of p-CREB and CBP to KLF4 needed for its transcription. Activation of adenylyl cyclase resulting from repression of autocrine C3ar1/C5ar1 signaling by upregulated CD55 concurrently led to p-CREB and CBP recruitment to KLF4-regulated genes, thereby conferring KLF4's transactivation. Accordingly, silencing CD55 in statin-treated HUVEC disabled CBP transfer from the E-selectin to the eNOS promoter. Importantly, silencing CD55 downregulated KLF4's expression. It did the same in untreated HUVEC transitioning from KLF4low growth to KLF4hi contact inhibition. KLF4's and CD55's function in ECs and macrophages thus are linked via a novel mechanism of gene transactivation. Because the two proteins are co-expressed in many cell types, CD55's activity may be broadly tied to KLF4's immunosuppressive and antithrombotic activities.

CD55 in cancer: Complementing functions in a non-canonical manner.

CD55, or decay accelerating factor, is a membrane lipid microdomain-associated, GPI-anchored protein implicated in the shielding of cells from complement-mediated attack via accelerating decay of C3 and C5. Loss of CD55 is associated with a number of pathologies due to hyperactivation of the complement system. CD55 is also implicated in cancer progression thought to be driven via its role in cell shielding mechanisms. We now appreciate that CD55 can signal intracellularly to promote malignant transformation, cancer progression, cell survival, angiogenesis, and inhibition of apoptosis. Outside-in signaling via CD55 is mediated by signaling pathways including JNK, JAK/STAT, MAPK/NF-κB, and LCK. Moreover, CD55 is enriched in the cancer stem cell (CSC) niche of multiple tumors including breast, ovarian, cervical, and can be induced by chemotherapeutics and hypoxic environments. CSCs are implicated in tumor recurrence and chemoresistance. Here, we review the unexpected roles of CD55 in cancer including the roles of canonical and noncanonical pathways that CD55 orchestrates. We will highlight opportunities for therapeutic targeting CD55 and gaps in the field that require more in-depth mechanistic insights.

Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα.

Background: Case reports are available showing that patients develop symptoms of acute arthritis during or after recovery from SARS-CoV-2 infection. Since the interrelation is still unknown, our aim was to study the impact of the SARS-CoV-2 nucleocapsid protein (NP) on human fibroblast-like synoviocytes and human endothelial cells (hEC) in terms of complement and cytokine regulation. Methods: Non-arthritic (K4IM) synoviocyte, arthritic (HSE) synoviocyte cell lines and primary hEC were stimulated with recombinant NP and/or TNFα. Analyses of cell viability, proliferation, gene and protein expression of cytokines and complement factors were performed. Results: NP suppressed significantly the vitality of hEC and proliferation of HSE. NP alone did not induce any significant changes in the examined gene expressions. However, NP combined with TNFα induced significantly higher TNFα in HSE and K4IM as well as higher IL-6 and CD55 gene expression in HSE and suppressed C3aR1 gene expression in hEC. HSE proliferated twice as fast as K4IM, but showed significantly lesser gene expressions of CD46, CD55, CD59 and TNFα with significantly higher IL-6 gene expression. CD35 gene expression was undetectable in K4IM, HSE and hEC. Conclusions: NP might contribute in combination with other inflammatory factors to complement regulation in arthritis.

The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa.

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CD55 is upregulated by cAMP/PKA/AKT and modulates human decidualization via Src and ERK pathway and decidualization-related genes.

Decidualization is an essential process for embryo implantation and maintenance of pregnancy, and abnormal decidualization contributed to several pregnancy disorders like a miscarriage. The objective of this study was to explore the regulation and function of CD55 in human decidualization. By immunohistochemical staining, it was found that CD55 expression was higher in first-trimester decidua than in the endometrium. In both primary endometrial stromal cells and immortalized cell line T-hESCs, CD55 was upregulated by induction of in vitro decidualization with medroxyprogesterone acetate (MPA) and 8-Br-cAMP. During decidualization in vitro, CD55 was stimulated by 8-Br-cAMP in a time- and concentration-dependent manner, which was reversed by a PKA inhibitor H89 and partially by an AKT activator SC79. Knocking down CD55 expression diminished the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1), accompanied by inhibition of Src, aberrant activation of ERK and decreased expression of several decidualization-related genes, including FOXO1, EGFR, and STAT3. Furthermore, the decidua of unexplained miscarriage women and the endometrium of unexplained infertile women both exhibited decreased CD55 expression. Collectively, these findings revealed that 8-Br-cAMP promotes CD55 expression via PKA activation and AKT dephosphorylation, and decreased CD55 impairs decidualization by inactivation of Src, aberrant activation of ERK pathway, and compromised expression of decidualization-related genes, indicating that CD55 deficiency may contribute to the pathogenesis of spontaneous miscarriage and infertility.

Endothelial-specific loss of Krüppel-Like Factor 4 triggers complement-mediated endothelial injury.

Thrombotic microangiopathy (TMA) in the kidney represents the most severe manifestation of kidney microvascular endothelial injury. Despite the source of the inciting event, the diverse clinical forms of kidney TMA share dysregulation of endothelial cell transcripts and complement activation. Here, we show that endothelial-specific knockdown of Krüppel-Like Factor 4 (Klf4)ΔEC, an anti-inflammatory and antithrombotic zinc-finger transcription factor, increases the susceptibility to glomerular endothelial injury and microangiopathy in two genetic murine models that included endothelial nitric oxide synthase knockout mice and aged mice (52 weeks), as well as in a pharmacologic model of TMA using Shiga-toxin 2. In all models, Klf4ΔEC mice exhibit increased pro-thrombotic and pro-inflammatory transcripts, as well as increased complement factors C3 and C5b-9 deposition and histologic features consistent with subacute TMA. Interestingly, complement activation in Klf4ΔEC mice was accompanied by reduced expression of a key KLF4 transcriptional target and membrane bound complement regulatory gene, Cd55. To assess a potential mechanism by which KLF4 might regulate CD55 expression, we performed in silico chromatin immunoprecipitation enrichment analysis of the CD55 promotor and found KLF4 binding sites upstream from the CD55 transcription start site. Using patient-derived kidney biopsy specimens, we found glomerular expression of KLF4 and CD55 was reduced in patients with TMA as compared to control biopsies of the unaffected pole of patient kidneys removed due to kidney cancer. Thus, our data support that endothelial Klf4 is necessary for maintenance of a quiescent glomerular endothelial phenotype and its loss increases susceptibility to complement activation and induction of prothrombotic and pro-inflammatory pathways.