RESEARCH ARTICLE CRITICAL CARE
PCSK9 is a critical regulator of the innate immune response and septic shock outcome Keith R. Walley,1* Katherine R. Thain,1 James A. Russell,1 Muredach P. Reilly,2 Nuala J. Meyer,3,4 Jane F. Ferguson,2 Jason D. Christie,3,4,5 Taka-aki Nakada,6 Chris D. Fjell,1 Simone A. Thair,1 Mihai S. Cirstea,1 John H. Boyd1 A decrease in the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9) increases the amount of lowdensity lipoprotein (LDL) receptors on liver cells and, therefore, LDL clearance. The clearance of lipids from pathogens is related to endogenous lipid clearance; thus, PCSK9 may also regulate removal of pathogen lipids such as lipopolysaccharide (LPS). Compared to controls, Pcsk9 knockout mice displayed decreases in inflammatory cytokine production and in other physiological responses to LPS. In human liver cells, PCSK9 inhibited LPS uptake, a necessary step in systemic clearance and detoxification. Pharmacological inhibition of PCSK9 improved survival and inflammation in murine polymicrobial peritonitis. Human PCSK9 loss-of-function genetic variants were associated with improved survival in septic shock patients and a decrease in inflammatory cytokine response both in septic shock patients and in healthy volunteers after LPS administration. The PCSK9 effect was abrogated in LDL receptor (LDLR) knockout mice and in humans who are homozygous for an LDLR variant that is resistant to PCSK9. Together, our results show that reduced PCSK9 function is associated with increased pathogen lipid clearance via the LDLR, a decreased inflammatory response, and improved septic shock outcome.
INTRODUCTION Microbial cell walls contain pathogenic lipid moieties such as lipopolysaccharide (LPS; Gram-negative bacteria), lipoteichoic acid (a structurally similar glycolipid found in Gram-positive bacteria) (1), and phospholipomannan (fungal pathogens) (2). These pathogen-associated lipids are major ligands for mammalian innate immune receptors [Tolllike receptors (TLRs)] and thus figure prominently in the septic inflammatory response (septic shock, or sepsis). Septic shock is an often fatal complication of a severe microbial infection (sepsis) that triggers an uncontrolled systemic inflammatory response (“cytokine storm”) and subsequent organ failure. Beyond antibiotic therapy, there are currently no effective treatments for sepsis or septic shock. Free pathogen lipids in the circulation are quickly bound by transfer proteins (3). Microbial LPS-binding protein (LBP) and bactericidal permeability-increasing protein (BPI) are similar to the endogenous mammalian lipid transfer proteins phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP), all of which bind pathogen lipids (3). When they are either bound to transfer proteins or free in the circulation, pathogen lipids trigger an inflammatory response through TLRs (4). In addition, when bound to transfer proteins (5), pathogen lipids are incorporated initially into high-density lipoproteins (HDLs) and subsequently can be transferred to low- or very-low-density lipoproteins (LDLs and VLDLs, respectively) (6), cleared from the blood by the liver, detoxified in the liver, and ultimately secreted in the bile (7). Binding, sequestering, and clearance from the blood reduces the in-
flammatory response induced by pathogen lipids (5, 7, 8). Therefore, understanding mechanisms of clearance from the blood for microbial lipids might provide the insight into the development of new therapeutic strategies for sepsis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulatory molecule that inhibits clearance of endogenous cholesterol lipid from the blood. PCSK9 binds the LDL receptor (LDLR) on hepatocytes, stimulates its internalization, promotes LDLR lysosomal degradation, and prevents recycling of LDLR to the cell surface (9–11). Thus, PCSK9 decreases LDLR cell surface density and, in turn, decreases clearance of LDL particles and increases plasma LDL cholesterol. Loss-offunction (LOF) variants of the PCSK9 gene result in increase clearance of LDL cholesterol from the circulation (12–18), whereas PCSK9 gain-offunction (GOF) variants have the opposite effect (13, 19) with a concordant and marked impact on a person’s risk of developing cardiovascular disease (20), coronary artery disease (12), and myocardial infarction (20, 21). These genetic discoveries triggered a race to develop PCSK9 inhibitors (22), which now show great promise in increasing LDL clearance (9) and lowering blood LDL cholesterol as well as the risk of atherosclerosis and its sequelae (20). We hypothesized that pathogen lipid clearance may also be modulated by PCSK9 inhibition, particularly in view of the striking similarities between mechanisms of transport and clearance of cholesterol and pathogen lipids (3, 5).
RESULTS 1 Centre for Heart Lung Innovation, University of British Columbia, Vancouver V6Z 1Y6, Canada. 2Cardiovascular Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 3Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 4 Center for Translational Lung Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 5Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 6Department of Emergency and Critical Care Medicine, Chiba University Graduate School of Medicine, Chiba 260-8677, Japan. *Corresponding author. E-mail: [email protected]
To test this hypothesis, we administered LPS intraperitoneally to Pcsk9 knockout (Pcsk9–/–) mice and genetic background control mice to measure LPS clearance, the cytokine inflammatory response, and clinically relevant physiological outcomes. Six hours after LPS administration, Pcsk9–/– mice had lower plasma concentrations of the early proinflammatory cytokine tumor necrosis factor a (TNFa) (P = 0.018); the integrative inflammatory cytokine interleukin-6 (IL-6), which is associated with survival in human sepsis (P = 0.004); the anti-inflammatory cytokine
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RESEARCH ARTICLE Fig. 1. Response to LPS in wild-type and Pcsk9–/– mice. Top: Plasma cytokine concentrations at baseline (top) and after LPS. A multiplex cytokine assay measured TNFa, IL-6, IL-10, JE, and MIP-2 at baseline and 6 hours after intraperitoneal injection of LPS (20 mg/kg). There were no significant differences at baseline shown in the bottom right frame (n = 9), whereas all cytokines were significantly (P < 0.05) lower in Pcsk9–/– mice (n = 9) than wild-type control mice (n = 9) after exposure to LPS. Bottom: (A) Activity phenotype. All 10 wild-type control mice had an activity index of 0 (no movement) by 6 hours after LPS administration compared to none of 10 Pcsk9–/– mice. Analysis of variance (ANOVA) demonstrates a difference between Pcsk9–/– and wild-type mice (P = 2.2 × 10–16) that was statistically significant (*P < 0.05) in hours 3 to 6. Typically, an activity index less than 0.5 represents a terminal state. (B) Body temperature. Wild-type control mice demonstrated a progressive loss of body temperature over the 6 hours after LPS administration such that 6 of the 10 mice had a body temperature 35°C) using a heating pad. A midline laparotomy (2 cm) was performed, and the cecum was exposed and ligated using a 6-0 suture anterior to the iliocecal valve, without causing intestinal obstruction. The cecum was perforated once through and through in the midsection using a 19-gauge needle. The incision was closed, and postoperatively, mice received 1 ml of normal saline subcutaneously. Beginning 6 hours postoperatively, mice received imipenem (25 mg/kg) subcutaneously twice daily. Sham operation included all of the above but did not puncture the cecum. Bacterial copy number in lung tissue assessed with real-time qPCR Lung tissue was rinsed in ice-cold phosphate-buffered saline (PBS) and snap-frozen in liquid nitrogen, followed by DNA extraction. Standard curves for reverse transcription qPCR (RT-qPCR) were generated as follows. Genomic DNA was extracted from E. coli DH5a bacteria grown in LB broth, using DNeasy kit (Qiagen, 69506). To construct a standard curve by qPCR, we compared cycle threshold versus copy number in which DNA is diluted 10-fold from 9.92 × 10–7 to 9.92 × 10–1 ng/ml (corresponding to 1,000,000 to 1 bacterial genomes). Real-time qPCR was performed on the ViiA 7 system (Applied Biosystems) for a 466–base pair (bp) fragment of the bacterial 16S ribosomal DNA, which was amplified using the forward primer 5′TCCTACGGGAGGCAGCAGT-3′ and the reverse primer 5′-GGACTACCAGGGTATCTAATCCTGTT-3′, as described in (47). Our published modifications to the published protocol included using a DNA-binding dye (SYBR Green) rather than the original TaqMan probe (28). The 466-bp product was amplified under the following conditions: 95°C for 5 min; 40 cycles of 95°C for 30 s, 60.5°C for 30 s, and 72°C for 60 s; and then a 10-min extension at 72°C. Bacterial copy number per milliliter was derived from the E. coli DH5a–derived standard curve. Double-blind, randomized controlled trial design for CLP A laboratory technician (M.S.C.) labeled packages of sterile 1-ml syringes (one package per mouse) with randomly generated numbers and filled them with 100 mg of PCSK9-blocking antibody (BPS Bioscience, 71207) or an IgG1 isotype control (R&D Systems, MAB002). An animal tech-
nician (Y. Wang) was blinded as to the identity of the syringes. Mice were injected once daily subcutaneously with the blinded solution beginning 6 hours after CLP. The team that analyzed the survival data was blinded to the identity of the syringes until assessment of the final mouse was completed. Berberine pretreatment to inhibit hepatic PCSK9 mRNA expression. At concentrations of 1 to 10 mM, berberine inhibits transcription of PCSK9 within 24 hours (34, 48). With a volume of distribution of 1 ml and a molecular weight of 371, we targeted plasma levels of 10 mM in mice by administering a bolus intraperitoneal injection of berberine, followed by maintenance infusion. Specifically, mice (body weight, 25 g) were injected intraperitoneally 60 hours before exposure to LPS, with 100 mg of berberine (Sigma-Aldrich) in 100 ml of sterile saline or saline vehicle. The next morning, a 72-hour osmotic pump (ALZET) with an infusion rate of 1 ml/hour was implanted intraperitoneally. Pumps contained 100 mg of berberine or vehicle saline. We confirmed that berberine pretreatment decreased hepatic expression of Pcsk9 using RT-PCR (Supplementary Methods). Ldlr knockout (Ldlr–/–) mice with and without inhibition of Pcsk9 expression. Twelve male Ldlr–/– mice (B6.129S7- Ldlrtm1Her/ J, Jackson Laboratories) 10 to 12 weeks of age and 25 g in weight were pretreated with berberine or sterile saline via a 72-hour infusion pump. Mice were injected intraperitoneally with LPS (100 mg/kg), and activity and body temperature were scored over the next 6 hours. In vitro assay of hepatocyte LPS clearance Immortalized human hepatocytes (HepG2 cell line, American Type Culture Collection) were seeded into a 24-well plate and grown to 80% confluence. The culture medium for the duration of the experiment was 80% DMEM (Invitrogen, 11965-065) and 20% human plasma from pooled healthy donors. Addition of PCSK9. Three hours before LPS treatment, cells were treated with purified recombinant human PCSK9 protein (3 mg/ml) (ACROBiosystems, PC9-H5223) or a vehicle control. Four and 19 hours after, LPS cells were again treated with recombinant human PCSK9 (3 mg/ml) or a vehicle control. Cells were treated with Alexa Fluor 488– conjugated LPS (Invitrogen, L-23351) or with standard nonfluorescent LPS (Sigma, L2880) as a control. After 24 hours of LPS treatment, each well was rinsed two times with PBS) and detached from the wells with Accutase (BD 561527). Cells were collected, washed with PBS, and resuspended in 500 ml of PBS, then analyzed by flow cytometry (Beckman Coulter Gallios Flow Cytometer). Cells were gated by forward and side scatter for viability using previously determined parameters; 20,000 gated cells were counted per sample. The output of interest was MFI from the instrument’s FL1 laser (excitation wavelength, 488 nm, emission wavelength, 525/20 nm). Background autofluorescence of cells treated with nonfluorescent LPS was subtracted to determine the fluorescence level resulting from uptake of the LPS conjugate. Data analysis was performed using Kaluza Analysis 1.3 software (Beckman Coulter). Human genetic association studies VASST derivation cohort. VASST was a multicenter, randomized, double-blind, controlled trial evaluating the efficacy of vasopressin versus norepinephrine in 778 patients who had septic shock (29) and vasopressor infusion of at least 5 mg/min of norepinephrine or equivalent (30). Inclusion criteria and clinical phenotyping are described elsewhere (30). DNA was available from 632 patients. The research ethics boards of all participating institutions approved this trial, and written
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RESEARCH ARTICLE informed consent was obtained from all patients or their authorized representatives. The research ethics board at the coordinating center (University of British Columbia) approved the genetic analysis. SPH validation cohort. All patients admitted to the intensive care unit (ICU) at SPH in Vancouver, Canada between July 2000 and January 2004 were screened, and of these, 415 patients were classified as having septic shock, had DNA available, and were successfully genotyped. Septic shock was defined by the presence of two or more diagnostic criteria for the systemic inflammatory response syndrome (29), proven or suspected infection, new dysfunction of at least one organ, and hypotension despite adequate fluid resuscitation (29). Inclusion criteria and clinical phenotyping are described elsewhere (8). The Institutional Review Board at SPH and the University of British Columbia approved the study. GENE population. Healthy, nonobese subjects (n = 294) with selfreported European or African American racial background were recruited for participation in the GENE study (49). Major exclusion criteria included medication or supplement use or evidence of any disease. After overnight acclimatization, LPS [E. coli–derived LPS (1 ng/kg); U.S. standard reference, lot no. CCRE-LOT-1+2, Clinical Center, Pharmacy Department at NIH, Bethesda, MD] was administered as an intravenous bolus, and subjects were monitored for 24 hours (49). Serial blood draws were taken (5 min before and 1, 2, 4, 6, 12, and 24 hours after LPS injection). Serum and plasma were obtained for measurement of cytokines, proteins, and lipids, and buffy coat was stored for genetic analyses. Samples were stored at –80°C before analysis. The protocol was approved by the University of Pennsylvania Institutional Review Board, had regulatory oversight by the U.S. Food and Drug Administration (LPS: IND 5984), and was monitored by an NIH-appointed data safety and monitoring board. Genotyping and cytokine measurements from these human cohorts are described in Supplementary Methods. Statistical analysis We used repeated-measures ANOVA to test for differences in activity level, temperature, and ejection fraction between Pcsk9–/– and wild-type control mice over time. We used unpaired t tests to test for differences in mean arterial pressure because this variable was measured using two different instruments at baseline and at 6 hours after LPS administration. Cytokine concentrations were log-normally distributed, so we tested for differences in logarithms of concentrations for murine TNFa, IL-6, IL-10, JE, and MIP-2 and for replication of directionally similar changes in human homologs of TNFa, IL-6, IL-10, MCP-1, and IL-8 using a one-way ANOVA. To assess changes in cytokine concentrations for the murine CLP model, we tested for differences in change from day 1 in log-transformed and normalized values using the slope coefficient estimates and SEs for the two treatment groups [analysis of covariance (ANCOVA)]. We tested SNPs for Hardy-Weinberg equilibrium using a c2 test. For human septic shock, our primary analysis of 28-day survival curves used a log-rank test stratified by Caucasian or non-Caucasian ancestry, and a secondary analysis used logistic regression to examine 28-day survival by PCSK9 genotype, including the covariates of age, gender, Caucasian ancestry, and a surgical versus medical primary diagnosis in the statistical model. Subgroup SNP analysis. Survival analysis up to 28 days was performed used Cox proportional hazards models for SNPs using the covariates age, gender, ancestry, and surgical diagnosis. We required a minimum of three patients for each ethnicity in each cohort; therefore,
we included only Caucasians, Asians, North American aboriginals, and Indians (patients of Latino or Middle Eastern ancestry were excluded because of low numbers). We performed the analysis for the VASST and SPH ICU1 cohorts independently as well as merged (for the merged data set, a cohort factor was included as a covariate). All SNP alleles were present in more than 1% of the patients except for the homozygous 2 (GG) for rs505151 in only two patients in the ICU1 cohort; to improve robustness of modeling, homozygous 2 for rs505151 was replaced with heterozygous 1 and interpreted as one or more variant alleles. Each SNP was evaluated independently using the numeric values 0, 1, and 2 for wild-type and 1 or 2 SNP variant bases present. An additional LOF variable representing any LOF SNP present with values 0 or 1 was also evaluated. The method coxph (http://www.R-project.org) from the base stats package in the R statistical language was used. To test LPS-evoked differences in plasma analytes, subjects were categorized as LOF carriers if they carried one or more minor alleles of the three LOF SNPs (rs562556, rs11583680, and rs11591147) and no copies of the minor allele for the GOF SNP rs505151. Peak IL-6 levels and area under the IL-6 curve, calculated using the trapezoidal rule, were compared between LOF and non-LOF by rank sum test. To allow adjustment for genetic ancestry and covariates shown to influence IL-6 measurements [gender, BMI, and age (49)], values were normalized, and linear regression including the first three components of the principal components analysis for genetic ancestry, gender, age, and BMI was performed. Differences were considered significant if P < 0.05. All analyses were performed using R (version 2.8.1, http://www.R-project. org), SPSS version 22.0 (SPSS Inc.), or Stata 12.0 statistical software packages.
SUPPLEMENTARY MATERIALS www.sciencetranslationalmedicine.org/cgi/content/full/6/258/258ra143/DC1 Methods Table S1. Physiological response of mice to LPS. Table S2. Allele frequency and Hardy-Weinberg equilibrium in VASST cohort. Table S3. Baseline characteristics of patients by PCSK9 LOF and GOF genotype. Table S4. Logistic regression. LOF and GOF effects on 28-day survival in septic shock cohorts.
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Bateman, K. R. Walley, Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kB dependent inflammatory response. Cardiovasc. Res. 72, 384–393 (2006). M. J. Scott, S. Liu, R. A. Shapiro, Y. Vodovotz, T. R. Billiar, Endotoxin uptake in mouse liver is blocked by endotoxin pretreatment through a suppressor of cytokine signaling-1– dependent mechanism. Hepatology 49, 1695–1708 (2009). T. A. Lagace, D. E. Curtis, R. Garuti, M. C. McNutt, S. W. Park, H. B. Prather, N. N. Anderson, Y. K. Ho, R. E. Hammer, J. D. Horton, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J. Clin. Invest. 116, 2995–3005 (2006). N. Lyle, J. Boyd, The potential for PCR based testing to improve diagnosis and treatment of sepsis. Curr. Infect. Dis. Rep. 15, 372–379 (2013). J. H. Boyd, J. A. Russell, C. D. Fjell, The meta-genome of sepsis: Host genetics, pathogens and the acute immune response. J. Innate Immun. 6, 272–283 (2014). American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit. Care Med. 20, 864–874 (1992). J. A. Russell, K. R. Walley, J. Singer, A. C. Gordon, P. C. Hébert, D. J. Cooper, C. L. Holmes, S. Mehta, J. T. Granton, M. M. Storms, D. J. Cook, J. J. Presneill, D. Ayers; VASST Investigators, Vasopressin versus norepinephrine infusion in patients with septic shock. N. Engl. J. Med. 358, 877–887 (2008). J. Mayne, T. C. Ooi, A. Raymond, M. Cousins, L. Bernier, T. Dewpura, F. Sirois, M. Mbikay, J. Davignon, M. Chrétien, Differential effects of PCSK9 loss of function variants on serum lipid and PCSK9 levels in Caucasian and African Canadian populations. Lipids Health Dis. 12, 70 (2013). A. K. Soutar, R. P. Naoumova, Mechanisms of disease: Genetic causes of familial hypercholesterolemia. Nat. Clin. Pract. Cardiovasc. Med. 4, 214–225 (2007). S. Rashid, D. E. Curtis, R. Garuti, N. N. Anderson, Y. Bashmakov, Y. K. Ho, R. E. Hammer, Y. A. Moon, J. D. Horton, Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc. Nat. Acad. Sci. U.S.A. 102, 5374–5379 (2005). J. Cameron, T. Ranheim, M. A. Kulseth, T. P. Leren, K. E. Berge, Berberine decreases PCSK9 expression in HepG2 cells. Atherosclerosis 201, 266–273 (2008). H. B. Xiao, Z. L. Sun, H. B. Zhang, D. S. Zhang, Berberine inhibits dyslipidemia in C57BL/6 mice with lipopolysaccharide induced inflammation. Pharmacol. Rep. 64, 889–895 (2012). F. Gao, H. E. Ihn, M. W. Medina, R. M. Krauss, A common polymorphism in the LDL receptor gene has multiple effects on LDL receptor function. Hum. Mol. Genetics 22, 1424–1431 (2013). K. Shioji, T. Mannami, Y. Kokubo, N. Inamoto, S. Takagi, Y. Goto, H. Nonogi, N. Iwai, Genetic variants in PCSK9 affect the cholesterol level in Japanese. J. Hum. Genet. 49, 109–114 (2004). S. N. Chen, C. M. Ballantyne, A. M. Gotto Jr., Y. Tan, J. T. Willerson, A. J. Marian, A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis. J. Am. Coll. Cardiol. 45, 1611–1619 (2005). A. Kumar, D. Roberts, K. E. Wood, B. Light, J. E. Parrillo, S. Sharma, R. Suppes, D. Feinstein, S. Zanotti, L. Taiberg, D. Gurka, A. Kumar, M. Cheang, Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34, 1589–1596 (2006). P. T. Kovanen, D. W. Bilheimer, J. L. Goldstein, J. J. Jaramillo, M. S. Brown, Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog. Proc. Nat. Acad. Sci. U.S.A. 78, 1194–1198 (1981). A. Pocathikorn, R. R. Taylor, C. D. Mamotte, Atorvastatin increases expression of low-density lipoprotein receptor mRNA in human circulating mononuclear cells. Clin. Exp. Pharmacol. Physiol. 37, 471–476 (2010). W. A. Flegel, M. W. Baumstark, C. Weinstock, A. Berg, H. Northoff, Prevention of endotoxininduced monokine release by human low- and high-density lipoproteins and by apolipoprotein A-I. Infect. Immun. 61, 5140–5146 (1993). V. Novack, J. MacFadyen, A. Malhotra, Y. Almog, R. J. Glynn, P. M. Ridker, The effect of rosuvastatin on incident pneumonia: Results from the JUPITER trial. CMAJ 184, E367–E372 (2012).
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RESEARCH ARTICLE 44. M. B. Rothberg, C. Bigelow, P. S. Pekow, P. K. Lindenauer, Association between statins given in hospital and mortality in pneumonia patients. J. Gen. Intern. Med. 27, 280–286 (2012). 45. P. S. Kruger, M. L. Harward, M. A. Jones, C. J. Joyce, K. M. Kostner, M. S. Roberts, B. Venkatesh, Continuation of statin therapy in patients with presumed infection: A randomized controlled trial. Am. J. Respir. Crit. Care Med. 183, 774–781 (2011). 46. K. R. Walley, N. W. Lukacs, T. J. Standiford, R. M. Strieter, S. L. Kunkel, Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect. Immun. 64, 4733–4738 (1996). 47. M. A. Nadkarni, F. E. Martin, N. A. Jacques, N. Hunter, Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148, 257–266 (2002). 48. H. Li, B. Dong, S. W. Park, H. S. Lee, W. Chen, J. Liu, Hepatocyte nuclear factor 1a plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine. J. Biol. Chem. 284, 28885–28895 (2009). 49. J. F. Ferguson, P. N. Patel, R. Y. Shah, C. K. Mulvey, R. Gadi, P. S. Nijjar, H. M. Usman, N. N. Mehta, R. Shah, S. R. Master, K. J. Propert, M. P. Reilly, Race and gender variation in response to evoked inflammation. J. Transl. Med. 11, 63 (2013). Funding: This work was supported by a Canadian Institutes of Health Research grant to K.R.W. and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, NIH, through grant UL1TR000003 as well as an NIH–National Heart, Lung, and Blood Institute Specialized Centers of Clinically Oriented Research Project grant (P50-HL-083799) to M.P.R. Author contributions: K.R.W. contributed to study conception, experimental design, and analysis and wrote the manuscript. K.R.T. assisted with experiment design, data collection, and analysis and contributed to manuscript writing. J.A.R. contributed
to project development and offered critical insight to manuscript content. M.P.R. conceived of and conducted all LPS administration experiments and contributed to manuscript writing. N.J.M. conducted the LPS administration analysis and contributed to manuscript writing. J.F.F. conducted the LPS administration experiments and reviewed the manuscript. J.D.C. contributed to LPS administration experiments and contributed to manuscript writing. T.N. contributed to human data analysis and manuscript critique. C.D.F. contributed to the statistical analysis. S.A.T. prepared the human samples for cytokine analysis and critiqued the manuscript. M.S.C. conducted cytokine and endotoxin assays and manuscript writing and editing. J.H.B. contributed to study conception, experimental design, analysis, and manuscript writing. Competing interests: The University of British Columbia has filed a provisional patent application covering aspects of this manuscript. K.R.W., J.H.B., and J.A.R. are listed as inventors. K.R.W., J.H.B., and J.A.R. have founded Cyon Therapeutics, which has licensed this intellectual property. The other co-authors declare that they have no competing interests. K.R.W. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Submitted 11 February 2014 Accepted 1 September 2014 Published 15 October 2014 10.1126/scitranslmed.3008782 Citation: K. R. Walley, K. R. Thain, J. A. Russell, M. P. Reilly, N. J. Meyer, J. F. Ferguson, J. D. Christie, T.-a. Nakada, C. D. Fjell, S. A. Thair, M. S. Cirstea, J. H. Boyd, PCSK9 is a critical regulator of the innate immune response and septic shock outcome. Sci. Transl. Med. 6, 258ra143 (2014).
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PCSK9 is a critical regulator of the innate immune response and septic shock outcome Keith R. Walley,1* Katherine R. Thain,1 James A. Russell,1 Muredach P. Reilly,2 Nuala J. Meyer,3,4 Jane F. Ferguson,2 Jason D. Christie,3,4,5 Taka-aki Nakada,6 Chris D. Fjell,1 Simone A. Thair,1 Mihai S. Cirstea,1 John H. Boyd1 A decrease in the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9) increases the amount of lowdensity lipoprotein (LDL) receptors on liver cells and, therefore, LDL clearance. The clearance of lipids from pathogens is related to endogenous lipid clearance; thus, PCSK9 may also regulate removal of pathogen lipids such as lipopolysaccharide (LPS). Compared to controls, Pcsk9 knockout mice displayed decreases in inflammatory cytokine production and in other physiological responses to LPS. In human liver cells, PCSK9 inhibited LPS uptake, a necessary step in systemic clearance and detoxification. Pharmacological inhibition of PCSK9 improved survival and inflammation in murine polymicrobial peritonitis. Human PCSK9 loss-of-function genetic variants were associated with improved survival in septic shock patients and a decrease in inflammatory cytokine response both in septic shock patients and in healthy volunteers after LPS administration. The PCSK9 effect was abrogated in LDL receptor (LDLR) knockout mice and in humans who are homozygous for an LDLR variant that is resistant to PCSK9. Together, our results show that reduced PCSK9 function is associated with increased pathogen lipid clearance via the LDLR, a decreased inflammatory response, and improved septic shock outcome.
INTRODUCTION Microbial cell walls contain pathogenic lipid moieties such as lipopolysaccharide (LPS; Gram-negative bacteria), lipoteichoic acid (a structurally similar glycolipid found in Gram-positive bacteria) (1), and phospholipomannan (fungal pathogens) (2). These pathogen-associated lipids are major ligands for mammalian innate immune receptors [Tolllike receptors (TLRs)] and thus figure prominently in the septic inflammatory response (septic shock, or sepsis). Septic shock is an often fatal complication of a severe microbial infection (sepsis) that triggers an uncontrolled systemic inflammatory response (“cytokine storm”) and subsequent organ failure. Beyond antibiotic therapy, there are currently no effective treatments for sepsis or septic shock. Free pathogen lipids in the circulation are quickly bound by transfer proteins (3). Microbial LPS-binding protein (LBP) and bactericidal permeability-increasing protein (BPI) are similar to the endogenous mammalian lipid transfer proteins phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP), all of which bind pathogen lipids (3). When they are either bound to transfer proteins or free in the circulation, pathogen lipids trigger an inflammatory response through TLRs (4). In addition, when bound to transfer proteins (5), pathogen lipids are incorporated initially into high-density lipoproteins (HDLs) and subsequently can be transferred to low- or very-low-density lipoproteins (LDLs and VLDLs, respectively) (6), cleared from the blood by the liver, detoxified in the liver, and ultimately secreted in the bile (7). Binding, sequestering, and clearance from the blood reduces the in-
flammatory response induced by pathogen lipids (5, 7, 8). Therefore, understanding mechanisms of clearance from the blood for microbial lipids might provide the insight into the development of new therapeutic strategies for sepsis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulatory molecule that inhibits clearance of endogenous cholesterol lipid from the blood. PCSK9 binds the LDL receptor (LDLR) on hepatocytes, stimulates its internalization, promotes LDLR lysosomal degradation, and prevents recycling of LDLR to the cell surface (9–11). Thus, PCSK9 decreases LDLR cell surface density and, in turn, decreases clearance of LDL particles and increases plasma LDL cholesterol. Loss-offunction (LOF) variants of the PCSK9 gene result in increase clearance of LDL cholesterol from the circulation (12–18), whereas PCSK9 gain-offunction (GOF) variants have the opposite effect (13, 19) with a concordant and marked impact on a person’s risk of developing cardiovascular disease (20), coronary artery disease (12), and myocardial infarction (20, 21). These genetic discoveries triggered a race to develop PCSK9 inhibitors (22), which now show great promise in increasing LDL clearance (9) and lowering blood LDL cholesterol as well as the risk of atherosclerosis and its sequelae (20). We hypothesized that pathogen lipid clearance may also be modulated by PCSK9 inhibition, particularly in view of the striking similarities between mechanisms of transport and clearance of cholesterol and pathogen lipids (3, 5).
RESULTS 1 Centre for Heart Lung Innovation, University of British Columbia, Vancouver V6Z 1Y6, Canada. 2Cardiovascular Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 3Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 4 Center for Translational Lung Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 5Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. 6Department of Emergency and Critical Care Medicine, Chiba University Graduate School of Medicine, Chiba 260-8677, Japan. *Corresponding author. E-mail: [email protected]
To test this hypothesis, we administered LPS intraperitoneally to Pcsk9 knockout (Pcsk9–/–) mice and genetic background control mice to measure LPS clearance, the cytokine inflammatory response, and clinically relevant physiological outcomes. Six hours after LPS administration, Pcsk9–/– mice had lower plasma concentrations of the early proinflammatory cytokine tumor necrosis factor a (TNFa) (P = 0.018); the integrative inflammatory cytokine interleukin-6 (IL-6), which is associated with survival in human sepsis (P = 0.004); the anti-inflammatory cytokine
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RESEARCH ARTICLE Fig. 1. Response to LPS in wild-type and Pcsk9–/– mice. Top: Plasma cytokine concentrations at baseline (top) and after LPS. A multiplex cytokine assay measured TNFa, IL-6, IL-10, JE, and MIP-2 at baseline and 6 hours after intraperitoneal injection of LPS (20 mg/kg). There were no significant differences at baseline shown in the bottom right frame (n = 9), whereas all cytokines were significantly (P < 0.05) lower in Pcsk9–/– mice (n = 9) than wild-type control mice (n = 9) after exposure to LPS. Bottom: (A) Activity phenotype. All 10 wild-type control mice had an activity index of 0 (no movement) by 6 hours after LPS administration compared to none of 10 Pcsk9–/– mice. Analysis of variance (ANOVA) demonstrates a difference between Pcsk9–/– and wild-type mice (P = 2.2 × 10–16) that was statistically significant (*P < 0.05) in hours 3 to 6. Typically, an activity index less than 0.5 represents a terminal state. (B) Body temperature. Wild-type control mice demonstrated a progressive loss of body temperature over the 6 hours after LPS administration such that 6 of the 10 mice had a body temperature 35°C) using a heating pad. A midline laparotomy (2 cm) was performed, and the cecum was exposed and ligated using a 6-0 suture anterior to the iliocecal valve, without causing intestinal obstruction. The cecum was perforated once through and through in the midsection using a 19-gauge needle. The incision was closed, and postoperatively, mice received 1 ml of normal saline subcutaneously. Beginning 6 hours postoperatively, mice received imipenem (25 mg/kg) subcutaneously twice daily. Sham operation included all of the above but did not puncture the cecum. Bacterial copy number in lung tissue assessed with real-time qPCR Lung tissue was rinsed in ice-cold phosphate-buffered saline (PBS) and snap-frozen in liquid nitrogen, followed by DNA extraction. Standard curves for reverse transcription qPCR (RT-qPCR) were generated as follows. Genomic DNA was extracted from E. coli DH5a bacteria grown in LB broth, using DNeasy kit (Qiagen, 69506). To construct a standard curve by qPCR, we compared cycle threshold versus copy number in which DNA is diluted 10-fold from 9.92 × 10–7 to 9.92 × 10–1 ng/ml (corresponding to 1,000,000 to 1 bacterial genomes). Real-time qPCR was performed on the ViiA 7 system (Applied Biosystems) for a 466–base pair (bp) fragment of the bacterial 16S ribosomal DNA, which was amplified using the forward primer 5′TCCTACGGGAGGCAGCAGT-3′ and the reverse primer 5′-GGACTACCAGGGTATCTAATCCTGTT-3′, as described in (47). Our published modifications to the published protocol included using a DNA-binding dye (SYBR Green) rather than the original TaqMan probe (28). The 466-bp product was amplified under the following conditions: 95°C for 5 min; 40 cycles of 95°C for 30 s, 60.5°C for 30 s, and 72°C for 60 s; and then a 10-min extension at 72°C. Bacterial copy number per milliliter was derived from the E. coli DH5a–derived standard curve. Double-blind, randomized controlled trial design for CLP A laboratory technician (M.S.C.) labeled packages of sterile 1-ml syringes (one package per mouse) with randomly generated numbers and filled them with 100 mg of PCSK9-blocking antibody (BPS Bioscience, 71207) or an IgG1 isotype control (R&D Systems, MAB002). An animal tech-
nician (Y. Wang) was blinded as to the identity of the syringes. Mice were injected once daily subcutaneously with the blinded solution beginning 6 hours after CLP. The team that analyzed the survival data was blinded to the identity of the syringes until assessment of the final mouse was completed. Berberine pretreatment to inhibit hepatic PCSK9 mRNA expression. At concentrations of 1 to 10 mM, berberine inhibits transcription of PCSK9 within 24 hours (34, 48). With a volume of distribution of 1 ml and a molecular weight of 371, we targeted plasma levels of 10 mM in mice by administering a bolus intraperitoneal injection of berberine, followed by maintenance infusion. Specifically, mice (body weight, 25 g) were injected intraperitoneally 60 hours before exposure to LPS, with 100 mg of berberine (Sigma-Aldrich) in 100 ml of sterile saline or saline vehicle. The next morning, a 72-hour osmotic pump (ALZET) with an infusion rate of 1 ml/hour was implanted intraperitoneally. Pumps contained 100 mg of berberine or vehicle saline. We confirmed that berberine pretreatment decreased hepatic expression of Pcsk9 using RT-PCR (Supplementary Methods). Ldlr knockout (Ldlr–/–) mice with and without inhibition of Pcsk9 expression. Twelve male Ldlr–/– mice (B6.129S7- Ldlrtm1Her/ J, Jackson Laboratories) 10 to 12 weeks of age and 25 g in weight were pretreated with berberine or sterile saline via a 72-hour infusion pump. Mice were injected intraperitoneally with LPS (100 mg/kg), and activity and body temperature were scored over the next 6 hours. In vitro assay of hepatocyte LPS clearance Immortalized human hepatocytes (HepG2 cell line, American Type Culture Collection) were seeded into a 24-well plate and grown to 80% confluence. The culture medium for the duration of the experiment was 80% DMEM (Invitrogen, 11965-065) and 20% human plasma from pooled healthy donors. Addition of PCSK9. Three hours before LPS treatment, cells were treated with purified recombinant human PCSK9 protein (3 mg/ml) (ACROBiosystems, PC9-H5223) or a vehicle control. Four and 19 hours after, LPS cells were again treated with recombinant human PCSK9 (3 mg/ml) or a vehicle control. Cells were treated with Alexa Fluor 488– conjugated LPS (Invitrogen, L-23351) or with standard nonfluorescent LPS (Sigma, L2880) as a control. After 24 hours of LPS treatment, each well was rinsed two times with PBS) and detached from the wells with Accutase (BD 561527). Cells were collected, washed with PBS, and resuspended in 500 ml of PBS, then analyzed by flow cytometry (Beckman Coulter Gallios Flow Cytometer). Cells were gated by forward and side scatter for viability using previously determined parameters; 20,000 gated cells were counted per sample. The output of interest was MFI from the instrument’s FL1 laser (excitation wavelength, 488 nm, emission wavelength, 525/20 nm). Background autofluorescence of cells treated with nonfluorescent LPS was subtracted to determine the fluorescence level resulting from uptake of the LPS conjugate. Data analysis was performed using Kaluza Analysis 1.3 software (Beckman Coulter). Human genetic association studies VASST derivation cohort. VASST was a multicenter, randomized, double-blind, controlled trial evaluating the efficacy of vasopressin versus norepinephrine in 778 patients who had septic shock (29) and vasopressor infusion of at least 5 mg/min of norepinephrine or equivalent (30). Inclusion criteria and clinical phenotyping are described elsewhere (30). DNA was available from 632 patients. The research ethics boards of all participating institutions approved this trial, and written
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RESEARCH ARTICLE informed consent was obtained from all patients or their authorized representatives. The research ethics board at the coordinating center (University of British Columbia) approved the genetic analysis. SPH validation cohort. All patients admitted to the intensive care unit (ICU) at SPH in Vancouver, Canada between July 2000 and January 2004 were screened, and of these, 415 patients were classified as having septic shock, had DNA available, and were successfully genotyped. Septic shock was defined by the presence of two or more diagnostic criteria for the systemic inflammatory response syndrome (29), proven or suspected infection, new dysfunction of at least one organ, and hypotension despite adequate fluid resuscitation (29). Inclusion criteria and clinical phenotyping are described elsewhere (8). The Institutional Review Board at SPH and the University of British Columbia approved the study. GENE population. Healthy, nonobese subjects (n = 294) with selfreported European or African American racial background were recruited for participation in the GENE study (49). Major exclusion criteria included medication or supplement use or evidence of any disease. After overnight acclimatization, LPS [E. coli–derived LPS (1 ng/kg); U.S. standard reference, lot no. CCRE-LOT-1+2, Clinical Center, Pharmacy Department at NIH, Bethesda, MD] was administered as an intravenous bolus, and subjects were monitored for 24 hours (49). Serial blood draws were taken (5 min before and 1, 2, 4, 6, 12, and 24 hours after LPS injection). Serum and plasma were obtained for measurement of cytokines, proteins, and lipids, and buffy coat was stored for genetic analyses. Samples were stored at –80°C before analysis. The protocol was approved by the University of Pennsylvania Institutional Review Board, had regulatory oversight by the U.S. Food and Drug Administration (LPS: IND 5984), and was monitored by an NIH-appointed data safety and monitoring board. Genotyping and cytokine measurements from these human cohorts are described in Supplementary Methods. Statistical analysis We used repeated-measures ANOVA to test for differences in activity level, temperature, and ejection fraction between Pcsk9–/– and wild-type control mice over time. We used unpaired t tests to test for differences in mean arterial pressure because this variable was measured using two different instruments at baseline and at 6 hours after LPS administration. Cytokine concentrations were log-normally distributed, so we tested for differences in logarithms of concentrations for murine TNFa, IL-6, IL-10, JE, and MIP-2 and for replication of directionally similar changes in human homologs of TNFa, IL-6, IL-10, MCP-1, and IL-8 using a one-way ANOVA. To assess changes in cytokine concentrations for the murine CLP model, we tested for differences in change from day 1 in log-transformed and normalized values using the slope coefficient estimates and SEs for the two treatment groups [analysis of covariance (ANCOVA)]. We tested SNPs for Hardy-Weinberg equilibrium using a c2 test. For human septic shock, our primary analysis of 28-day survival curves used a log-rank test stratified by Caucasian or non-Caucasian ancestry, and a secondary analysis used logistic regression to examine 28-day survival by PCSK9 genotype, including the covariates of age, gender, Caucasian ancestry, and a surgical versus medical primary diagnosis in the statistical model. Subgroup SNP analysis. Survival analysis up to 28 days was performed used Cox proportional hazards models for SNPs using the covariates age, gender, ancestry, and surgical diagnosis. We required a minimum of three patients for each ethnicity in each cohort; therefore,
we included only Caucasians, Asians, North American aboriginals, and Indians (patients of Latino or Middle Eastern ancestry were excluded because of low numbers). We performed the analysis for the VASST and SPH ICU1 cohorts independently as well as merged (for the merged data set, a cohort factor was included as a covariate). All SNP alleles were present in more than 1% of the patients except for the homozygous 2 (GG) for rs505151 in only two patients in the ICU1 cohort; to improve robustness of modeling, homozygous 2 for rs505151 was replaced with heterozygous 1 and interpreted as one or more variant alleles. Each SNP was evaluated independently using the numeric values 0, 1, and 2 for wild-type and 1 or 2 SNP variant bases present. An additional LOF variable representing any LOF SNP present with values 0 or 1 was also evaluated. The method coxph (http://www.R-project.org) from the base stats package in the R statistical language was used. To test LPS-evoked differences in plasma analytes, subjects were categorized as LOF carriers if they carried one or more minor alleles of the three LOF SNPs (rs562556, rs11583680, and rs11591147) and no copies of the minor allele for the GOF SNP rs505151. Peak IL-6 levels and area under the IL-6 curve, calculated using the trapezoidal rule, were compared between LOF and non-LOF by rank sum test. To allow adjustment for genetic ancestry and covariates shown to influence IL-6 measurements [gender, BMI, and age (49)], values were normalized, and linear regression including the first three components of the principal components analysis for genetic ancestry, gender, age, and BMI was performed. Differences were considered significant if P < 0.05. All analyses were performed using R (version 2.8.1, http://www.R-project. org), SPSS version 22.0 (SPSS Inc.), or Stata 12.0 statistical software packages.
SUPPLEMENTARY MATERIALS www.sciencetranslationalmedicine.org/cgi/content/full/6/258/258ra143/DC1 Methods Table S1. Physiological response of mice to LPS. Table S2. Allele frequency and Hardy-Weinberg equilibrium in VASST cohort. Table S3. Baseline characteristics of patients by PCSK9 LOF and GOF genotype. Table S4. Logistic regression. LOF and GOF effects on 28-day survival in septic shock cohorts.
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RESEARCH ARTICLE 44. M. B. Rothberg, C. Bigelow, P. S. Pekow, P. K. Lindenauer, Association between statins given in hospital and mortality in pneumonia patients. J. Gen. Intern. Med. 27, 280–286 (2012). 45. P. S. Kruger, M. L. Harward, M. A. Jones, C. J. Joyce, K. M. Kostner, M. S. Roberts, B. Venkatesh, Continuation of statin therapy in patients with presumed infection: A randomized controlled trial. Am. J. Respir. Crit. Care Med. 183, 774–781 (2011). 46. K. R. Walley, N. W. Lukacs, T. J. Standiford, R. M. Strieter, S. L. Kunkel, Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect. Immun. 64, 4733–4738 (1996). 47. M. A. Nadkarni, F. E. Martin, N. A. Jacques, N. Hunter, Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148, 257–266 (2002). 48. H. Li, B. Dong, S. W. Park, H. S. Lee, W. Chen, J. Liu, Hepatocyte nuclear factor 1a plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine. J. Biol. Chem. 284, 28885–28895 (2009). 49. J. F. Ferguson, P. N. Patel, R. Y. Shah, C. K. Mulvey, R. Gadi, P. S. Nijjar, H. M. Usman, N. N. Mehta, R. Shah, S. R. Master, K. J. Propert, M. P. Reilly, Race and gender variation in response to evoked inflammation. J. Transl. Med. 11, 63 (2013). Funding: This work was supported by a Canadian Institutes of Health Research grant to K.R.W. and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, NIH, through grant UL1TR000003 as well as an NIH–National Heart, Lung, and Blood Institute Specialized Centers of Clinically Oriented Research Project grant (P50-HL-083799) to M.P.R. Author contributions: K.R.W. contributed to study conception, experimental design, and analysis and wrote the manuscript. K.R.T. assisted with experiment design, data collection, and analysis and contributed to manuscript writing. J.A.R. contributed
to project development and offered critical insight to manuscript content. M.P.R. conceived of and conducted all LPS administration experiments and contributed to manuscript writing. N.J.M. conducted the LPS administration analysis and contributed to manuscript writing. J.F.F. conducted the LPS administration experiments and reviewed the manuscript. J.D.C. contributed to LPS administration experiments and contributed to manuscript writing. T.N. contributed to human data analysis and manuscript critique. C.D.F. contributed to the statistical analysis. S.A.T. prepared the human samples for cytokine analysis and critiqued the manuscript. M.S.C. conducted cytokine and endotoxin assays and manuscript writing and editing. J.H.B. contributed to study conception, experimental design, analysis, and manuscript writing. Competing interests: The University of British Columbia has filed a provisional patent application covering aspects of this manuscript. K.R.W., J.H.B., and J.A.R. are listed as inventors. K.R.W., J.H.B., and J.A.R. have founded Cyon Therapeutics, which has licensed this intellectual property. The other co-authors declare that they have no competing interests. K.R.W. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Submitted 11 February 2014 Accepted 1 September 2014 Published 15 October 2014 10.1126/scitranslmed.3008782 Citation: K. R. Walley, K. R. Thain, J. A. Russell, M. P. Reilly, N. J. Meyer, J. F. Ferguson, J. D. Christie, T.-a. Nakada, C. D. Fjell, S. A. Thair, M. S. Cirstea, J. H. Boyd, PCSK9 is a critical regulator of the innate immune response and septic shock outcome. Sci. Transl. Med. 6, 258ra143 (2014).
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