Immunological risk and complement genetic evaluations in early onset de novo thrombotic microangiopathy after living donor kidney transplantation: A Japanese multicenter registry.

BACKGROUND: Thrombotic microangiopathy (TMA) after kidney transplantation (KTx), particularly early onset de novo (dn) TMA, requires immediate interventions to prevent irreversible organ damage. This multicenter study was performed to investigate the allogeneic clinical factors and complement genetic background of dnTMA after KTx. METHODS: Perioperative dnTMA after KTx within 1 week after KTx were diagnosed based on pathological or/and hematological criteria at each center, and their immunological backgrounds were researched. Twelve aHUS-related gene variants were examined in dnTMA cases. RESULTS: Seventeen recipients (15 donors) were enrolled, and all dnTMA cases were onset within 72-h of KTx, and 16 of 17 cases were ABO incompatible. The implementation rate of pre-transplant plasmaphereses therapies were low, including cases with high titers of anti-A/anti-B antibodies. Examination of aHUS-related gene variants revealed some deletions and variants with minor allele frequency (MAF) in Japan or East Asian genome databases in genes encoding alternative pathways and complement regulatory factors. These variants was positive in 8 cases, 6 of which were positive in both recipient and donor, but only in one graft loss case. CONCLUSIONS: Although some immunological risks were found for dnTMA after KTx, only a few cases developed into TMA. The characteristic variations revealed in the present study may be novel candidates related to dnTMA in Japanese or Asian patients, but not pathogenic variants of aHUS. Future studies on genetic and antigenic factors are needed to identify factors contributing to dnTMA after KTx.

Complement biomarkers reflect the pathological status of neuromyelitis optica spectrum disorders.

Complement is involved in the pathogenesis of neuroimmune disease, but the detailed pathological roles of the complement pathway remain incompletely understood. Recently, eculizumab, a humanized anti-C5 monoclonal antibody, has been clinically applied against neuroimmune diseases such as myasthenia gravis and neuromyelitis optica spectrum disorders (NMOSD). Clinical application of eculizumab is also being investigated for another neuroimmune disease, Guillain-Barré syndrome (GBS). However, while the effectiveness of eculizumab for NMOSD is extremely high in many cases, there are some cases of myasthenia gravis and GBS in which eculizumab has little or no efficacy. Development of effective biomarkers that reflect complement activation in these diseases is therefore important. To identify biomarkers that could predict disease status, we retrospectively analyzed serum levels of complement factors in 21 patients with NMOSD and 25 patients with GBS. Ba, an activation marker of the alternative complement pathway, was elevated in the acute phases of both NMOSD and GBS. Meanwhile, sC5b-9, an activation marker generated by the terminal complement pathway, was elevated in NMOSD but not in GBS. Complement factor H (CFH), a complement regulatory factor, was decreased in the acute phase as well as in the remission phase of NMOSD, but not in any phases of GBS. Together, these findings suggest that complement biomarkers, such as Ba, sC5b-9 and CFH in peripheral blood, have potential utility in understanding the pathological status of NMOSD.

Early Elevation of Complement Factor Ba Is a Predictive Biomarker for Transplant-Associated Thrombotic Microangiopathy.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous reports suggest that TA-TMA is caused by complement activation by complement-related genetic variants; however, this needs to be verified, especially in adults. Here, we performed a nested case-control study of allo-HSCT-treated adults at a single center. Fifteen TA-TMA patients and 15 non-TA-TMA patients, matched according to the propensity score, were enrolled. Based on a previous report showing an association between complement-related genes and development of TA-TMA, we first sequenced these 17 genes. Both cohorts harbored several genetic variants with rare allele frequencies; however, there was no difference in the percentage of patients in the TA-TMA and non-TA-TMA groups with the rare variants, or in the average number of rare variants per patient. Second, we measured plasma concentrations of complement proteins. Notably, levels of Ba protein on Day 7 following allo-HSCT were abnormally and significantly higher in TA-TMA than in non-TA-TMA cases, suggesting that complement activation via the alternative pathway contributes to TA-TMA. All other parameters, including soluble C5b-9, on Day 7 were similar between the groups. The levels of C3, C4, CH50, and complement factors H and I in the TA-TMA group after Day 28 were significantly lower than those in the non-TA-TMA group. Complement-related genetic variants did not predict TA-TMA development. By contrast, abnormally high levels of Ba on Day 7 did predict development of TA-TMA and non-relapse mortality. Thus, Ba levels on Day 7 after allo-HSCT are a sensitive and prognostic biomarker of TA-TMA.

Disseminated gonococcal infection in a Japanese man with complement 7 deficiency with compound heterozygous variants: A case report.

RATIONALE: Complement deficiency are known to be predisposed to disseminated gonococcal infection (DGI). We herein present a case of DGI involving a Japanese man who latently had a complement 7 deficiency with compound heterozygous variants. PATIENT CONCERNS: A previously healthy 51-year-old Japanese man complained of sudden-onset high fever. Physical examination revealed various skin lesions including red papules on his trunk and extremities, an impetigo-like pustule on left forearm, and tendinitis of his right forefinger. DIAGNOSIS: Blood culture testing detected gram-negative cocci, which was confirmed to be Neisseria gonorrhoeae based on mass spectrometry and a pathogen-specific PCR test. INTERVENTIONS: Screening tests for underlying immunocompromised factors uncovered that complement activities (CH50) was undetectable. With a suspicion of a congenital complement deficiency, genetic analysis revealed rare single nucleotide variants in complement 7 (C7), including c.281-1G>T and a novel variant c.1454C>T (p.A485V). CH50 was normally recovered by adding purified human C7 to the patient's serum, supporting that the patient has C7 deficiency with compound heterozygous variants. OUTCOMES: Under a diagnosis of DGI, the patient underwent an antibiotic treatment with cefotaxime for a week and was discharged without any sequela. LESSONS: DGI is a rare sexually-transmitted infection that potentially induces systemic complications. Complement immunity usually defeats N. gonorrhoeae and prevents the organism from causing DGI. This case highlighted the importance of suspecting a complement deficiency when a person develops DGI.

Purification, Quantification, and Functional Analysis of Collectins.

Native and recombinant collectins are purified by using mannan-agarose and an anti-collectin antibody column. The use of sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies against human mannan-binding lectin (MBL) enables elucidation of the collectin concentration in the blood, serum, and plasma. The collectin sugar specificity is demonstrated by determining the concentration of saccharide required to inhibit sugar binding by 50% in a saccharide-binding assay. Biological analyses including the complement-dependent hemolysis test and several other methods are used to evaluate collectin.

[New Complement Therapeutics in Complement-Related Diseases].

The complement was named after "a complement", a protein molecule that supports an antibody. It was considered previously that the complement mainly participates in protecting against microbial infections. But later, as research on biological functions in complement activation advanced drastically, it was elucidated that the complement could be involved in the onset of various diseases. In 2007, eculizumab (ECZ), an anti-C5 (complement factor 5) monoclonal antibody, was approved as a drug for paroxysmal nocturnal hemoglobinuria (PNH) in the United States. In Japan, ECZ was approved for PNH and atypical hemolytic uremic syndrome (aHUS) in 2010 and 2013, respectively. The success of ECZ created an opportunity for drug companies to develop new therapeutics targeting the complement system; development of complement therapeutics is now a major venture of pharmaceutical companies worldwide. Here, I will provide an outline of the approved complement therapeutics and those that are in development and clinical trial phase currently.

Changes in Mannose-Binding Lectin and Collectin Kidney 1 Levels in Sepsis Patients With and Without Disseminated Intravascular Coagulation.

In sepsis, systemic coagulation activation frequently causes disseminated intravascular coagulation (DIC), and the uncontrolled activation of the complement system can induce multiple organ dysfunction and poor prognosis. This study aimed to examine the association of DIC with levels of collectin kidney 1 (CL-K1), a novel collectin of the complement system, and mannose-binding lectin (MBL), a classical-type collectin in patients with sepsis. We collected blood samples prospectively from adult patients with sepsis admitted to the intensive care unit (ICU) from day 1 (admission) to day 5. The CL-K1 and MBL levels were measured by enzyme-linked immunosorbent assay, and DIC was diagnosed by using a scoring algorithm. The correlation of CL-K1 and MBL levels with other coagulation markers was analyzed. There were 37 patients with DIC (DIC group) and 15 without DIC (non-DIC group). Compared to the non-DIC group, the DIC group had more severe conditions and higher mortality. During the 5 days after ICU admission, plasma CL-K1 levels were similar between the groups, but plasma MBL levels were significantly lower in the DIC group. Plasma CL-K1 levels were weakly correlated with prothrombin time, activated partial thromboplastin time, and antithrombin levels; plasma MBL levels were weakly correlated with fibrin/fibrinogen degradation product levels and DIC score. In conclusion, during the first 5 days of ICU admission, plasma CL-K1 levels were similar between the DIC and non-DIC groups. However, plasma MBL levels were lower in the DIC group compared to the non-DIC group, and the significance of this difference grew gradually over time.

Collectin Kidney 1 Plays an Important Role in Innate Immunity against Streptococcus pneumoniae Infection.

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway.

Three pentraxins C-reactive protein, serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1.

BACKGROUND: Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. METHODS: We used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. RESULTS: We demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. CONCLUSION: Our current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. GENERAL SIGNIFICANCE: CL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.

The collectins CL-L1, CL-K1 and CL-P1, and their roles in complement and innate immunity.

Both the complement system and collectins play important roles in our innate immune system. The collectins, which are characterized by their inclusion of a collagen-like region and a calcium-dependent carbohydrate recognition domain, are pattern recognition molecules and include the well characterized proteins mannan-binding lectin (MBL) and the surfactant proteins SP-A/-D. Collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1) and collectin placenta 1 (CL-P1) are the most recently discovered collectins. Although their function is still under investigation, accumulating information suggests that CL-L1, CL-K1 and CL-P1 play important roles in host defense by recognizing a variety of microorganisms and interacting with effector proteins, including complement components. The recent establishment of the existence of CL-K1 in the circulation in form of heteromeric complexes with CL-L1 (known as CL-LK) and its activation of the lectin pathway via MASPs, drew new attention in the complement biology, which was further strengthened by the observed interactions between CL-P1 and CRP-C1q-factor H or properdin. Deficiency of either CL-K1 or MASP-3 has been demonstrated in 3MC syndrome patients with developmental abnormalities, showing that lectin pathway components, regulation and/or activation are essential during the embryonic development; another feature that they most likely share CL-P1. Herein, we discuss the recent characteristics and roles of the collectins CL-L1, CL-K1 and CL-P1 in the complement system, in innate immunity and their possible association with disease development and pathogenesis.

Collectin CL-P1 utilizes C-reactive protein for complement activation.

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.

Collectin CL-LK Is a Novel Soluble Pattern Recognition Receptor for Mycobacterium tuberculosis.

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome.

BACKGROUND: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly(204)Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. RESULTS: In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca(2+) binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca(2+) during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. CONCLUSIONS: We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca(2+) binding during biosynthesis.

Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL.

BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2µ2.

BACKGROUND: Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS: We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in µ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS: We identified µ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2µ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS: Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2µ2. GENERAL SIGNIFICANCE: This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.

Elevated plasma CL-K1 level is associated with a risk of developing disseminated intravascular coagulation (DIC).

Collectin kidney 1 (CL-K1) is a recently identified collectin that is synthesized in most organs and circulates in blood. CL-K1 is an innate immune molecule that may play a significant role in host defense. As some collectins also play a role in coagulation, we hypothesized that an effect of CL-K1 may be apparent in disseminated intravascular coagulation (DIC), a gross derangement of the coagulation system that occurs in the setting of profound activation of the innate immune system. DIC is a grave medical condition with a high incidence of multiple organ failure and high mortality and yet there are no reliable biomarkers or risk factors. In our present study, we measured plasma CL-K1 concentration in a total of 659 specimens, including 549 DIC patients, 82 non-DIC patients and 27 healthy volunteers. The median plasma CL-K1 levels in these cohorts were 424, 238 and 245 ng/ml, respectively, with no significant difference in the latter two groups. The incidence of elevated plasma CL-K1 was significantly higher in the DIC patients compared to the non-DIC patients, resulting in an odds ratio of 1.929 (confidence interval 1.041-3.866). Infection, renal diseases, respiratory diseases, and cardiac diseases were more frequently observed in the DIC group than in the non-DIC group. In the DIC group, vascular diseases were associated with elevated plasma CL-K1 levels while age and acute illness had little effect on plasma CL-K1 levels. Independent of DIC, elevated plasma CL-K1 levels were associated with respiratory disease and coagulation disorders. These results suggest that specific diseases may affect CL-K1 synthesis in an organ dependent manner and that elevated plasma CL-K1 levels are associated with the presence of DIC. Further investigations in cohorts of patients are warranted. We propose that elevated plasma CL-K1 may be a new useful risk factor and possibly biomarker for the prediction of developing DIC.

Differential cell-protective function of two resveratrol (trans-3,5,4'-trihydroxystilbene) glucosides against oxidative stress.

Resveratrol (trans-3,5,4'-trihydroxystilbene; RSV), a natural polyphenol, exerts a beneficial effect on health and diseases. RSV targets and activates the NAD(+)-dependent protein deacetylase SIRT1; in turn, SIRT1 induces an intracellular antioxidative mechanism by inducing mitochondrial superoxide dismutase (SOD2). Most RSV found in plants is glycosylated, and the effect of these glycosylated forms on SIRT1 has not been studied. In this study, we compared the effects of RSV and two glycosyl RSVs, resveratrol-3-O-ß-d-glucoside (3G-RSV; polydatin/piceid) and resveratrol-4'-O-ß-d-glucoside (4'G-RSV), at the cellular level. In oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays, the antioxidant activity of 3G-RSV was comparable to that of RSV, whereas the radical-scavenging efficiency of 4'G-RSV was less than 50% of that of RSV. However, 4'G-RSV, but not 3G-RSV, induced SIRT1-dependent histone H3 deacetylation and SOD2 expression in mouse C2C12 skeletal myoblasts; as with RSV, SIRT1 knockdown blunted these effects. RSV and 4'G-RSV, but not 3G-RSV, mitigated oxidative stress-induced cell death in C2C12 cells and primary neonatal rat cardiomyocytes. RSV and 4'G-RSV inhibited C2C12 cell proliferation, but 3G-RSV did not. RSV was found in both the intracellular and extracellular fractions of C2C12 cells that had been incubated with 4'G-RSV, indicating that 4'G-RSV was extracellularly deglycosylated to RSV, which was then taken up by the cells. C2C12 cells did not deglycosylate 3G-RSV. Our results point to 4'G-RSV as a useful RSV prodrug with high water solubility. These data also show that the in vitro antioxidative activity of these molecules did not correlate with their ability to protect cells from oxidative stress-induced apoptosis.