Serum Amyloid A Stimulates PKR Expression and HMGB1 Release Possibly through TLR4/RAGE Receptors
Serum amyloid A (SAA) proteins are known to be surrogate markers of sepsis, but their pathogenic roles remain poorly elucidated. Here we provide evidence to support a possible role of SAA as a pathogenic mediator of lethal sepsis. In a subset of septic patients for which serum high mobility group box 1 (HMGB1) levels paralleled the clinical scores, some anti-HMGB1 antibodies detected a 12-kDa protein belonging to the SAA family. In contrast to the most abundant SAA1, human SAA induced double-stranded RNA-activated protein kinase R (PKR) expression and HMGB1 release in the wild-type, but not toll-like receptor 4/receptor for advanced glycation end products (TLR4/RAGE)-deficient, macrophages. Pharmacological inhibition of PKR phosphorylation blocked SAA-induced HMGB1 release, suggesting an important role of PKR in SAA-induced HMGB1 release. In animal models of lethal endotoxemia and sepsis, recombinant SAA exacerbated endotoxemic lethality, whereas SAA-neutralizing immunoglobulins G (IgGs) significantly improved animal survival. Collectively, these findings have suggested SAA as an important mediator of inflammatory diseases. Highlights of this study include: human SAA is possibly only expressed in a subset of septic patients; SAA induces HMGB1 release via TLR4 and RAGE receptors; SAA supplementation worsens the outcome of lethal endotoxemia; whereas SAA-neutralizing antibodies confer protection against lethal endotoxemia and sepsis.
Despite recent advances in antibiotic therapy and intensive care, sepsis remains a significant problem in critically ill patients with >225,000 victims in the U.S. alone. The pathogenesis of sepsis remains poorly understood, but is attributable to dysregulated immune responses orchestrated by innate immune cells including macrophages/monocytes (1). Macrophages/monocytes are equipped with various pattern recognition receptors (PRRs) (such as the toll-like receptors [TLRs] TLR2, TLR4 and TLR9), which can recognize various pathogen-associated molecular patterns (PAMPs) (such as bacterial lipoproteins, endotoxins and CpG-DNA) (2). Upon PRR-PAMP engagement, innate immune cells sequentially release early (for example, tumor necrosis factor [TNF], interleukin [IL]-1, interferon [IFN]-γ and cold-inducible RNA-binding protein [CIRP]) (3,4) and late (for example, nitric oxide [NO] or high mobility group box 1 [HMGB1]) proinflammatory mediators (5,6). If dysregulated, the excessive release of these late mediators adversely contributes to the pathogenesis of lethal sepsis (4,7, 8, 9).
In addition to stimulating macrophages/monocytes to release late proinflammatory mediators, early cytokines also alter the expression of liver-derived acute-phase proteins that similarly participate in the regulation of inflammatory responses. For instance, TNF, IL-1β and interferon (IFN)-γ induce the expression of serum amyloid A (SAA) in hepatocytes (10) and macrophages/monocytes (11), resulting in subsequent SAA secretion upon cleaving off the signal sequence. The human SAA family is comprised of multiple members, including the most abundant SAA1, and other isoforms such as SAA, SAA2α, SAA2β and SAA3. Members of the SAA family share >95–98% identity within species, with >75% sequence homology between human and rodents. During endotoxemia, circulating SAA levels are significantly elevated (up to 1,000-fold) within 16–24 h as a result of de novo expression of early cytokine inducers and subsequent synthesis and secretion of SAAs (12,13). Clinically, SAAs have been implicated as biomarkers in cardiovascular disorders (14), ulcerative colitis (15) and sepsis (16). Extracellular SAA signals via a family of receptors including the receptor for advanced glycation end products (RAGE) (17), TLR2 (18,19) and TLR4 (20) to activate NLRP3 inflammasome (21) and to induce various cytokines and chemokines (22, 23, 24, 25).
Previously, we demonstrated that a ubiquitous nuclear protein, HMGB1, is released from macrophages/monocytes in response to exogenous PAMPs (for example, lipopolysaccharide [LPS] and CpG-DNA) (6,26) or endogenous cytokines (for example, IFN-γ or CIRP) (4,27). The nucleus-to-cytoplasm translocation of HMGB1 is mediated by the STAT1-mediated acetylation of the HMGB1 nuclear-localization sequences (28). The extracellular HMGB1 release is regulated by caspase 1- and the double-stranded RNA-activated protein kinase R (PKR)-dependent inflammasome activation (29,30), pyroptosis (31) or necroptosis (32). For instance, pharmacological inhibition of PKR interaction with pyroptosome components (for example, apoptosis-associated speck protein [ASC]) by the 7-desacetoxy-6,7-dehydrogedunin (7DG) (31) results in the interruption of pyroptosis. Similarly, the suppression of PKR-mediated phosphorylation of necrosome components (for example, the death domain receptor-interacting protein 1 kinase [RIP1] and RIP3) by kinase inhibitors (for example, C16) (32) leads to the impairment of necroptosis. It was previously unknown, however, whether SAA can induce PKR expression to stimulate HMGB1 release.
In this study, we report a possibility that SAA was expressed only in a subset of septic patients and stimulated the expression of PKR and caused HMGB1 release in wild-type, but not in TLR4/RAGE-deficient, macrophages. Pharmacological inhibition of PKR phosphorylation inhibited SAA-induced HMGB1 release, and administration of SAA-neutralizing immunoglobulins G (IgGs) significantly improved animal survival in sepsis. Collectively, these findings have suggested a possible role of SAA as an important mediator for lethal inflammatory diseases.
Materials and Methods
Crude bacterial endotoxin (LPS, E. coli 0111:B4; catalog no. L4130), mouse anti-β-actin antibodies (catalog no. A1978) were Sigma-Aldrich products. Calcein-AM (catalog no. C3099), CM-DiI (catalog no. C7001), Dulbecco modified Eagle medium (DMEM) (catalog no. 11995-065), penicillin/streptomycin (catalog no. 15140-122), fetal bovine serum (FBS) (catalog no. 26140079) and Trypan blue (catalog no. 15250-061) were Invitrogen products. Anti-PKR antibody (catalog no. sc-6282) and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (catalog no. sc-2060) were Santa Cruz Biotechnology products. Anti-phosphorylated PKR antibody (T451; catalog no. 07-886) was a Millipore product. Rabbit anti-p-STAT1 (Y701; catalog no. 7649S) was a Cell Signaling product. Rabbit anti-TLR9 (catalog no. NBP1-76680) was a Novus Biologicals product. Recombinant human SAA (catalog no. 300-13) and SAA1 (catalog no. 300-53) were PeproTech products. HRP-conjugated donkey anti-rabbit IgG was a GE Healthcare product (catalog no. NA934). HMGB1-specific polyclonal antibodies were generated in rabbits as previously described (6). TLR2, TLR4 and RAGE KO mice and TLR2/RAGE and TLR4/RAGE-double KO mice on a C57BL/6 genetic background were maintained at The Feinstein Institute for Medical Research as previously described (33). SAA1/2 knockout mice were generated by targeted deletion of the exon 2 of both Saa1 and Saa2 genes as previously described (34), and the knockout mice were backcrossed into a C57BL/6 genetic background. Because the KO mice were derived from C57BL/6 mice, small colonies of wild-type C57BL/6 (The Jackson Laboratory) were maintained under the same conditions.
Preparation of Recombinant HMGB1
The cDNA encoding for rat HMGB1 was cloned onto a pCAL-n vector, and the recombinant calmodulin-binding protein (CBP)-tagged HMGB1 (rHMGB1) was expressed in E. coli BL21 (DE3) pLysS cells as previously described (6). The rHMGB1 containing an ∼3-kDa CBP tag (CBP-HMGB1 fusion protein, 33 kDa) was expressed in E. coli and purified to remove contaminating endotoxin by Triton X-114 extraction, as previously described (35).
Murine macrophage-like RAW 264.7 and human monocytic U937 cells were obtained from the American Type Culture Collection (ATCC). Primary peritoneal macrophages were isolated from Balb/C mice (Taconic; male, 7–8 wks, 20–25 g) at 2–3 d after intraperitoneal injection of 2 mL thioglycollate broth (4%), as previously described (36,37). RAW 264.7 macrophages, U937 monocytes and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Adherent macrophages or monocytes were gently washed with, and cultured in, DMEM before stimulating with LPS (0.5 µg/mL), HMGB1 (0.5 µg/mL) or human SAA, in the absence or presence of PKR inhibitors or SAA-neutralizing IgGs for 16 h. Subsequently, the cell-conditioned culture media were analyzed respectively for levels of HMGB1, nitric oxide and other cytokines by Western blotting analysis, the Griess reaction and cytokine antibodies arrays as previously described (38,39).
The levels of HMGB1 in the culture medium were determined by Western blotting analysis as previously described (6,27). The levels of total PKR, phosphorylated PKR (P-PKR, T451) or phosphorylated STAT1 (P-STAT1, Y701) in primary macrophage lysates were determined by Western blotting analysis with reference to β-actin. Briefly, equal amounts of cellular proteins were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat milk, the membrane was incubated with respective antibodies overnight [anti-PKR, 1:250; anti-phospho-PKR (T451), 1:1,000; anti-P-STAT1 (Y701), 1:500; anti-β-actin, 1:5,000]. Subsequently, the membrane was incubated with the appropriate secondary antibody, and the immunoreactive bands were visualized by chemiluminescence technique.
Nitric Oxide Assay
The levels of NO in the culture medium were determined indirectly by measuring the NO2− production with a colorimetric assay on the basis of the Griess reaction (36,40). NO2− concentrations were determined with reference to a standard curve generated with sodium nitrite at various dilutions.
Cytokine Antibody Array
Human Cytokine Antibody Array C3 (catalog no. AAH-CYT-3-4) and Murine Cytokine Antibody Arrays (catalog no. M0308003, RayBiotech Inc., which respectively detect 42 and 62 cytokines on one membrane, were used to determine cytokine levels in human serum or cell culture medium as previously described (36,40). Briefly, the membranes were sequentially incubated with equal volumes of human serum (10 µL, mixed with 90 µL buffer) or cell culture medium (200 µL), primary biotin-conjugated antibodies and horseradish peroxidase-conjugated streptavidin. After exposing to X-ray film, the relative signal intensity was determined by using the Scion Image software.
Clinical Characterization of Septic Patients
As per the approval by the Feinstein Institute for Medical Research institutional review board ethics committee, blood samples (10 mL) were collected at various time points (0, 12, 24, 48 and 72 h) after the diagnosis of patients with septic shock, severe sepsis or sepsis, in the Department of Emergency Medicine, North Shore University Hospital. The American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference definitions of sepsis and septic shock were used for the diagnosis of these patients (41). As controls, blood samples (10 mL) were also collected from eight healthy individuals. Serum samples were analyzed for levels of HMGB1 and 42 other cytokines by Western blotting and cytokine antibody arrays, respectively.
MALDI-TOF Mass Spectrometry
To identify the 12-kDa band that crossreacted with anti-HMGB1 antibodies, serum samples were resolved by SDS-PAGE, and the corresponding 12-kDa band was subjected to MALDI-TOF mass spectrometry analysis. Briefly, the 12-kDa band was excised from the SDS-PAGE gel and subjected to in-gel trypsin digestion. The mass of the tryptic peptides was measured by matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF-MS) and then subjected to peptide mass fingerprinting database analysis to identify the 12-kDa protein (“P12”) in septic patients.
Analysis of Contaminating Bacterial Products
The recombinant human SAA and SAA1 were analyzed for contaminating bacterial products (such as lipoproteins, CpG-DNAs) by SDS-PAGE, followed by Coomassie blue or ethidium bromide staining. Recombinant HMGB1 protein was used as a control for comparison. Recombinant SAAs were tested for LPS content by the chromogenic Limulus amebocyte lysate assay (Endochrome; Charles River), and the endotoxin content was expressed as nanograms endotoxin per microgram of SAAs.
Animal Models of Endotoxemia and Polymicrobial Sepsis
This study was approved and performed in accordance with the guidelines for the care and use of laboratory animals at the Feinstein Institute for Medical Research, Manhasset, New York. To evaluate the role of SAA in lethal sepsis, Balb/C mice (male, 7–8 wks, 20–25 g) were subjected to lethal endotoxemia or sepsis induced by cecal ligation and puncture (CLP) as previously described (38,39,42). Briefly, the cecum of Balb/C mice was ligated at 5.0 mm from the cecal tip and then punctured once with a 22-gauge needle. Recombinant SAA or SAA-neutralizing antibodies were intraperitoneally administered into endotoxemic or septic mice at indicated doses and time points, and animal survival rates were monitored for up to 2 wks.
Generation of Anti-SAA Polyclonal Antibodies
Female New Zealand white rabbits were repetitively immunized with recombinant human SAA in combination with the Freund’s complete adjuvant, and blood was collected on 3-wk cycles of immunization and bleed. The antibody titers were determined by direct SAA ELISA, and total IgGs were purified from the serum using a Protein A affinity column as previously described (6). Briefly, rabbit serum was prebuffered with PBS and slowly loaded to a protein A column to allow sufficient binding of IgGs. After washing with 1× PBS to remove nonbound serum components, the IgGs were eluted with acidic buffer (0.1 mol/L glycine-HCl, pH 2.8) and then immediately dialyzed into 1× PBS buffer at 4°C overnight.
Peptide Dot Blotting
A library of 19 overlapping peptides (18-mer, offsite by 6) corresponding to the human SAA sequence were synthesized and spotted (1.0 µg in 2.5 µL) onto nitrocellulose membrane (Thermo Scientific, catalog no. 88013). Subsequently, the membrane was probed with IgGs from different rabbits following a standard protocol.
Data are expressed as mean ± SEM of two independent experiments in triplicates (n = 2). One-way analyses of variance (ANOVAs) followed by the Tukey’s test for multiple comparisons were used to compare between different groups. The Kaplan-Meier method was used to compare the differences in mortality rates between groups. A P value <0.05 was considered statistically significant.
Identification of Human SAA, but not SAA1, as an HMGB1 Inducer
Requirement of TLR4/RAGE Receptors in SAA-Induced HMGB1 Release
Requirement of TLR4/RAGE Receptors for SAA-Induced PKR Upregulation
Characterizing SAA-Neutralizing Polyclonal Antibodies
Roles of SAA in Animal Models of Lethal Endotoxemia and Sepsis
Despite high homology between human SAA and SAA1, their capacities in inducing HMGB1 release are dramatically different. The infrequently expressed SAA, unlike the most abundant SAA1, significantly induced HMGB1 release in TLR4/RAGE- and PKR-dependent mechanisms. These novel findings were consistent with several studies that echoed their dramatic differences in stimulating other cytokines/chemokines (22, 23, 24, 25). Although SAA contained minute amount of endotoxins, this trivial contamination was not likely the underlying cause for SAA-mediated HMGB1 release, because (a) Ultrapure LPS (free from bacterial proteins and nucleic acids; catalog # tlrl-pelps, InvivoGen) fails to trigger HMGB1 release even when given up to 10 µg/mL (29,30); (b) the similarly prepared SAA1 that contained comparable amounts of endotoxin still failed to induce HMGB1 release; (c) endotoxin-neutralizing agent (for example, polymyxin B) effectively abrogated LPS-induced (0.5 µg/mL), but not SAA-induced (0.5 µg/mL), HMGB1 release; and (d) the disruption of TLR4 receptor impaired crude LPS-, but not SAA-induced, HMGB1 release. Finally, consistent with the capacity of HDL in capturing SAA (52) and blocking its chemokine activities (53), we found that HDL (catalog no. L8039; Sigma-Aldrich) effectively attenuated SAA-induced HMGB1 release (data not shown), further supporting SAA as an inducer of HMGB1 release.
Both genetic and pharmacological approaches are routinely used to assess the divergent roles of inflammatory mediators in sepsis. However, caution should be exercised when using gene knockout approaches to evaluate the pathogenic roles of any particular mediators that are still critically needed for maintaining beneficial physiological functions. For instance, despite the well-established pathogenic role of HMGB1 in infection- and injury-elicited inflammatory diseases (60,61), the disruption of HMGB1 expression adversely renders animals more susceptible to infectious (62) or injurious insults (63,64), reinforcing the dramatically distinct roles of HMGB1 in health and disease (65). Similarly, in an animal model of dextran sodium sulfate (DSS)-induced colitis, SAA1/2 knockout mice appeared to be more susceptible to colitis, possibly because the intestinal epithelia-derived SAAs are still critically needed for bactericidal activities (34). Thus, we used pharmacological approaches to evaluate the role of SAA in animal models of lethal systemic inflammation. Consistent with the notion that early PAMP-elicited early inflammatory responses might still be needed for the innate immunity against infection (66), it was previously shown that prophylactic treatment with two anti-SAA IgGs (mc29 and mc4; 1:1, beginning at 12 h before CLP) reduced animal survival rates from 60% to 20% (67). In the present study, we found that delayed and repetitive administration of SAA-neutralizing polyclonal antibodies conferred significant protection against lethal sepsis, suggesting a pathogenic role of SAA during a late stage of sepsis. Consistently, after lethal endotoxemia (10 mg/kg), SAA1/SAA2 knockout mice exhibited a higher lethality (ratio of animal death: KO/WT = 6/4) within 24 h, followed by a lower mortality (KO/WT = 2/5) between 24 and 48 h, supporting a pathogenic role of SAAs during a late stage of sepsis.
In septic patients (68), multiple SAA isoforms have been found including the most abundant SAA1, as well as several scarce variants such as SAA, SAA2α and SAA2β. The SAA2s differ from each other only at position 71 (H versus R), but differ from SAA1 at seven to eight other positions, indicating a possible polymorphism (69). Indeed, the frequencies of the α and β alleles at the SAA2 locus varied among different populations (for example, the Turkish versus the Azerbaijan and Kazakh) (70). At present, it is not yet known why human SAA was only detected in a subset of septic patients. Although human SAA and SAA1 differ from each other only at position 60 (D versus N) and 71 (H versus R), it will be important to investigate whether a possible polymorphism is associated with SAA and SAA1. This is important because genetic polymorphisms of proinflammatory cytokines (for example, TNF) can influence their circulating levels and consequently influence the outcomes of sepsis (71).
Here we provided some evidence to support the possibility that SAA might be expressed only in a subset of septic patients. Unlike the most abundant SAA1, SAA significantly upregulated PKR and stimulated HMGB1 release in a TLR4/RAGE-dependent fashion. Pharmacological inhibition of PKR phosphorylation inhibited SAA-induced HMGB1 release, supporting an important role of PKR in SAA-induced HMGB1 release and NO production. In animal models of lethal endotoxemia and experimental sepsis, recombinant SAA exacerbated LPS-induced animal lethality, whereas SAA-neutralizing IgGs significantly improved animal survival. Collectively, these findings have suggested a possible role of SAA as an important mediator and potential therapeutic target for lethal inflammatory diseases.
W Li, KJ Tracey and H Wang are coinventors on a patent application entitled “SAA domain-specific antibodies and peptide antagonists and use thereof to treat inflammatory diseases.”
The authors thank Dr. Maria de Beer for providing the SAA1/SAA2 KO mice. This work was supported by the National Institute of General Medical Sciences (NIGMS, R01GM063075) and the National Center of Complementary and Alternative Medicine (NCCAM, R01AT05076).
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