Rachel E. Simmonds

Clarification of the Risk for Venous Thrombosis Associated with Hereditary Protein S Deficiency by Investigation of a Large Kindred with a Characterized Gene Defect

Vitamin K-dependent protein S plays an important role in the regulation of the coagulation cascade [1]. This protein increases the rate of degradation of activated factors V and VIII by acting as a cofactor to activated protein C, thereby limiting thrombin production. The presence of factor V, with which protein S acts in synergy, amplifies the function of protein S as a cofactor to activated protein C in factor VIII proteolysis. Protein S has recently been found to have anticoagulant functions independent of activated protein C: It directly inhibits procoagulant enzyme complexes [2, 3]. The relative importance of these different functions in maintaining intravascular fluidity is still unknown. However, heterozygous deficiency of protein S was described as a cause of venous thrombosis in 1984 [4] and has subsequently been identified in numerous clinically affected families, in which it is inherited as an autosomal dominant trait. This deficiency has been found in 1.5% to 7% of selected groups of thrombophilic patients [5-8], although an estimate of the prevalence in the general population awaits a sufficiently large study. Homozygous protein S deficiency is an extremely rare and life-threatening disorder associated with severe neonatal purpura fulminans [9]. Unlike other coagulation inhibitors, protein S has some functions that are affected by C4b-binding protein, a component of the complement cascade, to which 60% to 70% of protein S is bound in vivo [10]. Once bound, protein S can no longer act as a cofactor to activated protein C but retains some of its inhibitory properties. Because of the difference in function between the bound and unbound forms of protein S and uncertainty over which function is the most important, levels of both total and free protein S antigen are usually measured in patient plasma samples. The function of protein S as a cofactor to activated protein C may also be assessed. Protein S deficiency is diagnosed if one or more of these measurements is found to be below the lower limit of a laboratory reference range. Several problems are encountered in the diagnosis of protein S deficiency, including the large overlap in antigen levels between normal and heterozygous persons [11]. This overlap may be due to the effect of sex and hormones on total protein S levels [12, 13]. We recently described an age-related increase in total protein S antigen, independent of the influence of sex, in both normal and protein S-deficient persons [14]. This phenomenon also complicates diagnosis made on the basis of total protein S measurement and causes phenotypic variation within the same kindred. Furthermore, some assays for protein S activity are influenced by a mutation in the gene for factor V (Arg506Gln), which causes resistance to activated protein C and is a common, if relatively mild, risk factor for thrombosis. Heterozygosity for this mutation can result in apparent reduction of protein S cofactor activity to activated protein C in the laboratory assessment of normal persons [15]. Despite these problems with diagnosis, some studies have attempted to compute the risk for thrombosis associated with phenotypic protein S deficiency [11, 16-18]. In deficient families, the probability that affected family members remain thrombosis-free at 45 years of age has been reported to be 0.35 to 0.50 [11, 16]. However, the incidence of thrombosis varies among different families; this suggests problems with precise diagnosis or the presence of other genetic risk factors. Of note, no study has examined the risk associated with genetically confirmed protein S deficiency, which would remove the diagnostic uncertainties. The identification of gene mutations that cause protein S deficiency is complicated by the size of the gene (>80 kilobase-pairs) that encodes protein S [19-21] and by the presence of a pseudogene. However, an increasing number of studies have identified such mutations in probands or small family groups. The first database of protein S gene mutations was recently published by the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis [22]. Mutations in the coding region or at intron-exon boundaries have generally been identified in approximately 50% of protein S-deficient probands. We recently identified a single causative mutation (which results in a Gly295Val substitution) in a large protein S-deficient kindred [14]. This mutation is not thought to be common in the general population. The availability of comprehensive phenotypic, genotypic, and clinical data has enabled the interrelations among these data to be investigated and has thereby provided quantitative information on the risk for thrombosis associated with a mutation in the protein S gene and the value of different assays in predicting clinical events. Methods Participants The manifestations of thrombosis in the 122-member family under investigation (most family members live in northern Sweden) were attributed to protein S deficiency in 1993 [23]. This kindred has also been part of a larger study that involved 18 families with phenotypic protein S deficiency [16, 24] and provided an explanation for phenotypic variation in familial protein S deficiency [14]. All participants gave informed consent, and the medical ethics committee at the University of Lund approved all of these studies, including the present one. Study participants answered a questionnaire about their medical history, with emphasis on manifestations of deep venous thrombosis, pulmonary embolism, superficial thrombophlebitis, and arterial thrombosis. Symptomatic family members were also interviewed by a physician or had their medical records reviewed. The term deep venous thrombosis includes deep venous thrombosis of the leg and thrombosis in such unusual locations as the axillary, mesenteric, and cerebral veins. Thrombotic event refers to deep venous thrombosis, pulmonary embolism, or superficial thrombophlebitis diagnosed by a physician on the basis of physical examination. Laboratory Methods Blood sampling and routine coagulation were performed as described elsewhere [25]. Total and free protein S antigen levels were measured by doing radioimmunoassay [26]. Protein S levels were compared with laboratory reference ranges for levels of both free (reference range, 56 to 182 nmol/L) and total (reference range, 219 to 407 nmol/L) antigen. Persons receiving anticoagulation were compared with an anticoagulated control group. The control groups that we used have been described elsewhere [24]. Of the 122 family members, 44 had free protein S antigen levels below the lower limit of the reference range; 13 of the 44 were receiving oral anticoagulants at the time of sampling. Molecular Genetic Investigation The methods used to identify and detect the novel protein S gene mutation, Gly295Val, in 122 genomic DNA samples have been reported elsewhere [14, 27]. All 44 family members with reduced free protein S antigen levels were heterozygous for the mutation; the remaining 78 relatives who had normal free protein S antigen levels were normal at this site. This finding confirmed that the Gly295Val mutation was the cause of protein S deficiency in this family. A single asymptomatic family member with normal free protein S antigen levels had previously been found to be heterozygous for the factor V Arg506Gln mutation. Statistical Analysis Thrombosis-free survival curves were constructed according to the method of Kaplan and Meier [28]. Two curves were compared by using the log-rank test, which results in a test statistic with chi-squared distribution and one degree of freedom [29]. This analysis was performed by using Statistica software (Statsoft, Inc., Tulsa, Oklahoma). Univariate and multivariate Cox regression analyses [30] were performed by using Statistica software or SPS (SPS, Inc., Chicago, Illinois). All 122 family members were included in the analysis for risk for thrombosis. Results Demographic and Clinical Data Samples of plasma and genomic DNA were available for 122 germline family members (60 men and 62 women; mean age SD, 36 17 years [range, 7 to 82 years]) spanning five generations. The distribution of patient samples was 1, 8, 41, 57, and 15 from the first, second, third, fourth, and fifth generations, respectively. A histogram of the current ages of the study participants is shown in Figure 1. Twenty-five (57%) of the 44 family members with the Gly295Val mutation had one or more venous thrombotic events (mean age at first event, 31 years [range, 11 to 71 years]) compared with 5 (6%) of the 78 family members who lacked the mutation (mean age at first event, 29 years [range, 16 to 43 years]). The clinical manifestations in symptomatic family members are summarized in Table 1. First thrombotic events were associated with one or more circumstantial risk factors in 12 symptomatic relatives (48%) with the Gly295Val mutation and 2 family members (40%) who lacked the mutation (Table 2). Three carriers of the mutation (6.8%) and none of the normal family members had arterial thrombotic events (postoperative bilateral arterial thrombosis requiring bilateral above-the-knee amputation, myocardial infarction, and embolization requiring below-the-knee amputation). Figure 1. Histogram of the current ages of the investigated persons in the study family. Table 1. Clinical Manifestations of Venous Thrombosis in Symptomatic Family Members with and without the Gly295Val Mutation Table 2. Circumstantial Risk Factors Associated with First Thrombotic Episodes in 25 Symptomatic Relatives with the Gly295Val Mutation and 5 Symptomatic Relatives without the Mutation According to Kaplan-Meier analysis of thrombosis-free survival, the probability that a family member who carries the Gly295Val mutation would remain free of venous thrombosis at 30 years of age is 0.5 (95% CI, 0.33 to 0.66) compared with 0.97 (CI, 0.93 to 1.0) for normal family members (Figure 2)

Inhibitors of TLR8 Reduce TNF Production from Human Rheumatoid Synovial Membrane Cultures

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.

Mycolactone Inhibits Monocyte Cytokine Production by a Posttranscriptional Mechanism

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1β, IL-6, IL-10, and IFN-γ-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-κB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactones observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Recent advances: role of mycolactone in the pathogenesis and monitoring of Mycobacterium ulcerans infection/Buruli ulcer disease

Infection of subcutaneous tissue with Mycobacterium ulcerans can lead to chronic skin ulceration known as Buruli ulcer. The pathogenesis of this neglected tropical disease is dependent on a lipid‐like toxin, mycolactone, which diffuses through tissue away from the infecting organisms. Since its identification in 1999, this molecule has been intensely studied to elucidate its cytotoxic and immunosuppressive properties. Two recent major advances identifying the underlying molecular targets for mycolactone have been described. First, it can target scaffolding proteins (such as Wiskott Aldrich Syndrome Protein), which control actin dynamics in adherent cells and therefore lead to detachment and cell death by anoikis. Second, it prevents the co‐translational translocation (and therefore production) of many proteins that pass through the endoplasmic reticulum for secretion or placement in cell membranes. These pleiotropic effects underpin the range of cell‐specific functional defects in immune and other cells that contact mycolactone during infection. The dose and duration of mycolactone exposure for these different cells explains tissue necrosis and the paucity of immune cells in the ulcers. This review discusses recent advances in the field, revisits older findings in this context and highlights current developments in structure‐function studies as well as methodology that make mycolactone a promising diagnostic biomarker.

Pleiotropic molecular effects of the Mycobacterium ulcerans virulence factor mycolactone underlying the cell death and immunosuppression seen in Buruli ulcer

Mycolactone is a polyketide macrolide lipid-like secondary metabolite synthesized by Mycobacterium ulcerans, the causative agent of BU (Buruli ulcer), and is the only virulence factor for this pathogen identified to date. Prolonged exposure to high concentrations of mycolactone is cytotoxic to diverse mammalian cells (albeit with varying efficiency), whereas at lower doses it has a spectrum of immunosuppressive activities. Combined, these pleiotropic properties have a powerful influence on local and systemic cellular function that should explain the pathophysiology of BU disease. The last decade has seen significant advances in our understanding of the molecular mechanisms underlying these effects in a range of different cell types. The present review focuses on the current state of our knowledge of mycolactone function, and its molecular and cellular targets, and seeks to identify commonalities between the different functional and cellular systems. Since mycolactone influences fundamental cellular processes (cell division, cell death and inflammation), getting to the root of how mycolactone achieves this could have a profound impact on our understanding of eukaryotic cell biology.

Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone

ABSTRACT The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterise Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans. In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterise the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome–nascent-chain–Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61α subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the molecular mechanisms of Sec61-dependent protein translocation. Summary: The Buruli ulcer toxin mycolactone inhibits both the co- and post-translational pathways for protein translocation across the endoplasmic reticulum by perturbing the function of the Sec61 translocon.

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

TLR signalling and adapter utilization in primary human in vitro differentiated adipocytes.

Toll‐like receptors (TLRs) are central to innate immunity and yet their expression is widespread and not restricted to professional inflammatory cells. TLRs have been reported on adipocytes and have been implicated in obesity‐associated pathologies such as diabetes. Why TLRs are found on adipocytes is not clear although one hypothesis is that they may coordinate energy utilization for the energy intensive process of an immune response. We have explored TLR signalling in primary human in vitro differentiated adipocytes and investigated the specific adapter molecules that are involved. Only lipopolysaccharide (LPS), poly(I:C), Pam3CSK4 and MALP‐2 could induce the production of IL‐6, IL‐8 and MCP‐1 by adipocytes. Poly(I:C) alone caused a strong induction of type I interferons, as assessed by IP‐10 production. Using siRNA, it was confirmed that LPS‐dependent signalling in adipocytes occurs via TLR4 utilizing the adapter molecules MyD88, Mal and TRIF and caused rapid degradation of IκBα. Pam3CSK4 signalling utilized TLR2, MyD88 and Mal (but not TRIF). However, the response to poly(I:C) observed in these cells appeared not to require TRIF, but MyD88 was required for induction of NFκB‐dependent cytokines by Poly(I:C). Despite this, IκBα degradation could not be detected in poly(I:C) stimulated adipocytes at any time‐point up to 4 h. Indeed, IL‐6 transcription was not induced until 8–16 h after exposure. These data suggest that Pam3CSK4 and LPS signal via the expected routes in human adipocytes, whereas poly(I:C)/TLR3 signalling may act via a TRIF‐independent, MyD88‐dependent route.

Mycolactone reveals substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

ABSTRACT Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression that are characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, the translocation of TMP lumenal domains driven by an N-terminal cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation that is driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs. Highlighted Article: The exotoxin mycolactone interferes with the biogenesis of the majority of transmembrane proteins and its actions highlight differences in how distinct classes of these proteins initially engage the Sec61 translocon.