Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A

Edward J. Burgess, Laura R. Hoyt, Matthew J. Randall, Madeleine M. Mank, Joseph J. Bivona III, Philip L. Eisenhauer, Jason W. Botten, Bryan A. Ballif, Ying-Wai Lam, Matthew J. Wargo, Jonathan E. Boyson, Jennifer L. Ather and Matthew E. Poynter
J Immunol October 15, 2018, 201 (8) 2377-2384; DOI: https://doi.org/10.4049/jimmunol.1800503

Abstract

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli–derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell–derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

Introduction

The serum amyloid A (SAA) family of acute-phase proteins have been studied for decades as robust biomarkers for a wide array of inflammatory and autoimmune disorders as well as for their contribution to AA amyloidosis. Increasing ∼1000-fold in the serum in response to infection and injury, SAA proteins are evolutionarily conserved and are the major acute-phase proteins in vertebrates (1). Multiple isoforms of SAA are expressed in the liver (the largest source of acute-phase reactants) as well as in hematopoietic and nonhematopoietic cells throughout the body. SAA1 and SAA2 are highly homologous and predominantly produced by the liver, whereas SAA3 is an acutely expressed isoform produced in nonprimate mammals (2), and the constitutively expressed SAA4 does not increase in response to infection or injury (3).

Given its rapid and robust increase under inflammatory and autoimmune conditions, it has long been speculated that SAA is a mediator of the inflammatory process. Specifically, we and others have reported that the proinflammatory effects of SAA are largely mediated through the activation of TLR2 (4–18). However, the Escherichia coli–derived recombinant form of human SAA that is almost uniformly used by investigators (the apolipoprotein form of human SAA1 [apoSAA], a lipid-binding apolipoprotein that is a constituent of plasma lipoprotein) elicits strong proinflammatory responses not shared with the endogenous form of SAA. Specifically, although recombinant apoSAA promotes neutrophil activation and proinflammatory cytokine production, human plasma containing highly elevated levels of SAA displays neither of these effects (19, 20). Furthermore, transgenic overexpression of mouse (21) or human (20, 22) SAA1 in mice to levels that recapitulate those in the circulation of humans does not elicit a proinflammatory state. These results imply that further characterization of the differences in proinflammatory activities between recombinant and endogenous SAA is warranted.

SAA proteins associate with high-density lipoprotein (HDL), displacing human apolipoprotein A1 (apoA1) as the predominant apolipoprotein during inflammation (1, 23). Interestingly, the capacity of SAA to stimulate cells via TLR2 has been reported to be only when it is not incorporated into HDL (21). SAA has been shown to prevent the entry of enveloped viruses into cells (24–26) and reported to opsonize Gram-negative bacteria (27, 28). The outer membrane protein A (OmpA) of E. coli has been reported to bind SAA1, enabling it to augment the ability of neutrophils to ingest invading bacteria (27). Furthermore, SAA functions as a circulating chaperone for the small, lipophilic vitamin A derivative retinoic acid (29) and can associate with phospholipids to form larger particles (30). As SAA clearly has the capacity to associate with lipid-rich particles and compounds, we hypothesized that other lipophilic molecules, such as bacterial lipopeptides, may also associate with SAA. Bacterial lipopeptides are potent activators of TLR2, whereby they induce the production of proinflammatory cytokines and other effects that have been attributed to recombinant forms of SAA produced in E. coli. The objective of our studies was to compare the effects of E. coli–derived and eukaryotic cell–derived SAA1 proteins on TNF-α production from macrophages, the stimulation of TLR2, and the induction of Th17 responses, as well as to examine the contribution of bacterial lipoproteins to the capacity to induce the aforementioned proinflammatory effects.

Materials and Methods

Reagents

Chemicals were from Thermo Fisher Scientific (Hampton, NH) unless noted otherwise. Recombinant human apoSAA, recombinant human SAA1 (hSAA1), and apoA1, all made in E. coli, were from PeproTech (Rocky Hill, NJ). Recombinant mouse SAA1 made in E. coli was from R&D Systems (Minneapolis, MN). hSAA1 made in human embryonic kidney (HEK) cells was from OriGene (Rockville, MD). Ultrapure E. coli O111:B4 LPS and Pam3CSK4 were from InvivoGen (San Diego, CA). Plasmid pcDNA3 was from Invitrogen (Carlsbad, CA). pcDNA3.1+ encoding human SAA1 was from GenScript (Piscataway, NJ). Plasmid pCMV6-XL5 encoding human SAA1 was from OriGene. E. coli transformed with pLX304 plasmids encoding human TLR1 and TLR2 were from the DNASU Plasmid Repository (Tempe, AZ). Plasmids from these bacteria were purified using EndoFree Plasmid Kits from Qiagen (Germantown, MD). Polyethylenimine (PEI) was from Polysciences (Warrington, PA). Mini-PROTEAN TGX precast 4–20% polyacrylamide gels were from Bio-Rad (Hercules, CA). Immobilon nitrocellulose transfer membranes and Amicon Ultra centrifugal filter units with a 3-kDa molecular mass cutoff were from EMD Millipore (Billerica, MA). Lipoprotein lipase (LPL) from Pseudomonas sp. and OVA were from Sigma-Aldrich (St. Louis, MO). TOP10 chemically competent E. coli were from Invitrogen. Anti-CD3 and anti-CD28 Abs were from BD Biosciences (San Jose, CA).

Cells

Peripheral blood was collected from normal adult human donors under the approval of the University of Vermont Institutional Review Board (protocol no. M10-171). For the preparation of PBMCs, blood collected into EDTA-coated tubes was diluted 1:1 in HBSS (without calcium or magnesium), layered over lymphocyte separation medium (LSM; MP Biomedicals, Santa Ana, CA), and centrifuged at 500 × g for 30 min at room temperature. The top layer of plasma was aspirated and discarded, leaving 2–3 mm above the buffy coat, and the buffy coat and half the lower LSM layer were aspirated, mixed with HBSS, and washed twice. Neutrophils were enriched by resuspending the pellet obtained during the LSM preparation of PBMCs in 20 ml of HBSS and adding 20 ml of 3% dextran in HBSS, which was subsequently mixed by inversion several times. Erythrocytes were allowed to settle for 20 min at room temperature, after which the neutrophil-rich supernatant was transferred to a new tube and washed with HBSS. Erythrocytes were lysed by two rounds of resuspending the pellet in 10 ml of hypotonic lysis buffer for 30 s followed by the addition of an equal volume of re-equilibration buffer and washing. Splenocytes and bone marrow were collected from C57BL/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME). Studies were approved by the University of Vermont’s Institutional Animal Care and Use Committee (protocol no. 12-018) in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health, and efforts were made to minimize suffering. Sodium pentobarbital was administered via i.p. injection for euthanasia, and cells were processed as described (4). Bone marrow–derived dendritic cells (DCs) were generated as previously described (31), and CD4+ T cells were isolated from splenocytes by negative selection (STEMCELL Technologies, Vancouver, BC, Canada) (32). RAW 264.7, J774A.1, and HEK293T cells from American Type Culture Collection (Manassas, VA) were maintained in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies), 1% l-glutamine (Life Technologies), and 1× Primocin (InvivoGen). For experiments in which cell supernatants were examined by ELISA, cells were plated at 2.5 × 105 cells per well in 250 μl of media in a 48-well plate and allowed to grow overnight. The following day, the cells were treated as indicated within the figure legends for each experiment. Cell supernatants were harvested at the end of each experiment, spun down at 3300 × g for 10 min to pellet cellular debris, transferred to new tubes, and frozen at −20°C until analysis. For transfection, 5.6 × 104 HEK293T cells were seeded per square centimeter in each well of 6- or 96-well plates (for expression of SAA or TLR1/2, respectively) and transfected 1 d later with 11.1 μl/cm2 DMEM containing 0.2 μg/cm2 plasmid and 0.9 μg/cm2 of PEI per well.

Cytokine and SAA quantitation

Cell supernatants were analyzed for mouse TNF-α, IL-1β, or IL-4 using ELISA kits from BD Biosciences. Human IL-8, IL-6, TNF-α, IL-1β, and SAA1 as well as mouse IL-5, IL-13, IL-17A, and IFN-γ were measured by ELISA using DuoSet reagents from R&D Systems. All ELISAs were performed according to manufacturer’s instructions.

Fast protein liquid chromatography

Two hundred and fifty micrograms of apoSAA was run on a Superdex 75 size-exclusion column (GE Healthcare Life Sciences, Pittsburgh, PA) in PBS (pH 7.2) running buffer, quantitated for total protein (OD280) as it eluted from the column (90 fractions of ∼330 μl each were collected), and compared with the elution time of protein molecular mass standards to estimate its size. Briefly, a standard curve was generated by plotting the elution volume parameter (Kav) of several standards purchased from GE Healthcare (RNase A, aprotinin, carbonic anhydrase, OVA, and conalbumin) versus their log molecular masses. The Kav of SAA was read from this curve to estimate its molecular mass (∼76 kDa). Kav was calculated as follows: blue dextran was used to calculate the void volume (Vo) of the column, and the total bed volume (Vt) was 24 ml. Kav = (Elution volume (Ve) − Vo) / (Vt − Vo).

Mass spectrometry analysis

For in-gel digestion and subsequent analyses, apoSAA (50 μg) in Laemmli buffer with 5% 2-ME was separated on a 4–20% Tris-glycine gel at 140 V for 1 h 15 min. The gel was then stained in 30% Coomassie Brilliant Blue (40% methanol, 20% acetic acid, and 0.1% Brilliant Blue R [Sigma-Aldrich]) (diluted in destain) overnight, destained in 30% methanol and 20% acetic acid, and imaged using a CanoScan 8800F scanner (Canon, Melville, NY), and a cut map was created to divide the lanes into 12 specific cuts. Proteins within the gel slices were reduced in 25 mM DTT for 30 mins, alkylated in-gel in 10 mM iodoacetamide for 45 mins, and subjected to two rounds of dehydration with acetonitrile and rehydration with water prior to a final dehydration in acetonitrile. To the dry gel slices, 25 μl of 12 ng/μl sequence-grade trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate was added. The samples were placed on ice for 30 min and digested at 37°C overnight. Tryptic peptides were extracted with 2.5% formic acid in 50% acetonitrile while spinning in a microcentrifuge at 13,000 rpm for 10 min. The supernatant was collected, and the gel slices were dehydrated by twice incubating with 100% acetonitrile and collecting all the extractions from a given gel slice in the same tube, and solvent was removed using a vacuum centrifuge at 37°C. The peptides were resuspended in 2.5% acetonitrile and 2.5% formic acid, loaded using a Micro AS autosampler (Thermo Fisher Scientific, Pittsburgh, PA), and separated in a microcapillary column packed with 12 cm of Magic C18 200-Å 5-μm material (Michrom Bioresources, Auburn, CA). The peptides were separated and eluted with a 5–35% acetonitrile (0.15% formic acid) gradient using a Surveyor Plus HPLC pump instrument (Thermo Fisher Scientific) over 40 min, after a 15-min isocratic loading at 2.5% acetonitrile and 0.15% formic acid. Mass spectra were acquired in an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific) using 10 tandem mass spectrometry scans following each survey scan over the entire run. The human and E. coli International Protein Index forward and reverse concatenated databases were queried with SEQUEST software requiring a 2-Da precursor mass tolerance, tryptic peptide matches, +57.02 Da on cysteine residues, and allowing +15.99 Da for oxidation of methionine residues. Using cross-correlation and delta correlation scores, peptide matches were filtered to a false discovery rate of <0.01%, and when proteins were required to have at least three peptides identified, there were no remaining hits matching to the reverse database. For in-solution digestion and subsequent analyses, recombinant proteins (6 μg) were concentrated using a Savant SpeedVac concentrator (Thermo Fisher Scientific) and then reconstituted in 50 mM ammonium bicarbonate buffer containing 4% acetonitrile. Proteins were reduced in 100 mM DTT for 1 h at 56°C, alkylated in 200 mM iodoacetamide for 45 min at room temperature, and concentrated using a SpeedVac. Protein digestion was performed by reconstitution in digestion solution containing 100 mM ammonium bicarbonate, 4% acetonitrile, and 6 ng/μl mass spectrometry–grade trypsin (Promega) via incubation at 37°C for 18 h. Digestion was terminated with the addition of 10% formic acid, and samples were processed by ZipTip C18 P10 (EMD Millipore). Liquid chromatography–mass spectrometry-based protein identification was performed on a linear ion trap LTQ Orbitrap Discovery mass spectrometer coupled to a Surveyor MS Pump Plus (Thermo Fisher Scientific). Tryptic peptides were loaded onto a 100-μm × 120-mm capillary column packed with MAGIC C18 (5-μm particle size, 20-nm pore size; Michrom Bioresources) at a flow rate of 500 nl/min. Separated peptides were introduced into the linear ion trap via a nanospray ionization source and a laser-pulled ∼3-μm orifice with a spray voltage of 1.8 kV. Standard “top-ten” data-dependent acquisition was used, in which an Orbitrap survey scan from m/z 360–1600 at 30,000 resolution was paralleled by 10 collision-induced dissociation tandem mass spectrometry scans of the most abundant ions in the LTQ. Product ion spectra were searched in a target-decoy fashion using SEQUEST on Proteome Discoverer 1.4 (Thermo Fisher Scientific) against a curated E. coli database with apoSAA, human SAA1 and apoA1, and mouse SAA1 sequences incorporated. Search parameters were as follows: 1) full trypsin enzymatic activity; 2) two missed cleavages; 3) peptides between molecular masses of 350–5000 Da; 4) mass tolerance at 20 parts/million for precursor ions and at 0.8 Da for fragment ions; and 5) dynamic modifications on methionine (+15.99 Da: oxidation) and static modification on cysteine (+57.02 Da: carbamidomethylation). Filters were applied to limit the false positive rates to <1% in the data sets. Assigned protein gene names and aliases were compared with the UniProt KnowledgeBase (33) and E. coli Protein Database (34) databases, and the EcoTopic “LipoProteome” of EcoGene (35) was used to identify verified and probable E. coli lipoproteins. Data from the mass spectrometry analysis are included in Supplemental Table I.

Statistics

Data were analyzed by one-way ANOVA and Dunnett or Tukey multiple comparisons tests, or by two-way ANOVA and Tukey multiple comparisons test using GraphPad Prism 7.04 for Windows (GraphPad Software, La Jolla, CA.). A posttest-corrected p value smaller than 0.05 was considered statistically significant.

Results

E. coli–derived recombinant SAA proteins induce proinflammatory cytokine production and Th17 polarization

To compare the proinflammatory effects of SAA from different sources, we purchased recombinant SAA proteins from a number of vendors. apoSAA represents the most-commonly used recombinant form of SAA and was able to dose-dependently induce IL-8, IL-6, TNF-α, and IL-1β production from human PBMCs (Fig. 1A–D) and IL-8 from human neutrophils (Fig. 1E). Because apoSAA is human SAA1 made in E. coli but in which aa 60 and 71 are from SAA2, we also stimulated human PBMCs and neutrophils with human SAA1 also made from E. coli by the same vendor. Like apoSAA, hSAA1 induced robust proinflammatory cytokine production, whereas human SAA1 produced in HEK cells did not induce proinflammatory cytokine production from human PBMCs and neutrophils (Fig. 1A–E). Using TNF-α as an indicator of proinflammatory cytokine production, similar results were seen in primary mouse splenocytes (Fig. 1F) as well as the RAW (Fig. 1G) and J774 (Fig. 1H) mouse macrophage cell lines. Consequently, subsequent studies were conducted using J774 cells. apoSAA has been reported by us (4, 36) and others (11, 17) to activate the Nlrp3 inflammasome to induce IL-1β secretion. Whereas the addition of apoSAA or the synthetic bacterial lipopeptide Pam3CSK4 to LPS-primed J774 macrophages augmented IL-1β secretion, E. coli–derived mouse SAA1 and HEK-derived human SAA1 did not (Fig. 1I). A dose-response study in J774 macrophages revealed that the E. coli–derived human SAA1 proteins elicited maximal TNF-α production at 1 μg/ml, whereas the mouse SAA1 produced in E. coli (mSAA1) and HEK-derived human SAA1 did not induce TNF-α production even at concentrations as high as 10 μg/ml (Fig. 1J). apoSAA was subsequently separated into 90 fractions using size-exclusion chromatography (fast protein liquid chromatography [FPLC]). The fractions with the highest abundance of protein eluted at a time equivalent to a ∼72-kDa standard (Fig. 1K), suggesting that native SAA exists as a hexamer, as has been previously reported (37). We also found that only those fractions containing abundant quantities of SAA were capable of eliciting TNF-α production (Fig. 1L). These results suggest that the stimulatory capacity of E. coli–derived SAA is associated with the native protein and cannot be physically separated by size-exclusion chromatography.

FIGURE 1.
FIGURE 1.

Recombinant SAA produced in E. coli, but not produced from eukaryotic cells, stimulates proinflammatory cytokine production. Primary human PBMCs (A–D) and neutrophils (polymorphonuclear cells [PMNs]) (E) were unstimulated (control) or stimulated with 62, 250, or 1000 ng/ml of SAA from different sources for 24 (PBMC) or 3 h (PMN), after which cytokine concentrations were measured by ELISA. Primary C57BL/6J mouse splenocytes (F), mouse RAW macrophage cells (G), and J774 mouse macrophages (H) were unstimulated (control) or stimulated with 1 μg/ml of SAA from different sources. Culture supernatants were collected 24 h later and TNF-α concentrations were measured by ELISA. J774 mouse macrophages were unstimulated (control) or stimulated for 24 h with 100 ng/ml of LPS, 1 μg/ml of apoSAA, LPS plus 100 ng/ml of the lipopeptide Pam3CSK4 (Pam), LPS plus apoSAA, or LPS plus 1 μg/ml of human SAA1 made in HEK cells, and IL-1β concentrations in culture supernatants were measured by ELISA (I). J774 mouse macrophages were unstimulated (control) or stimulated for 24 h with 10, 100, or 1000 ng/ml of SAA from different sources, and TNF-α concentrations in culture supernatants were measured 24 h later by ELISA (J). Two hundred and fifty micrograms of apoSAA was separated by FPLC using a size-exclusion column, and 90 fractions were collected (K). Fractions obtained from FPLC were diluted 1:10 in serum-free media and used to stimulate J774 cells for 24 h, after which TNF-α concentrations were measured by ELISA (L). Data are mean ± SEM and are representative of three independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett test for multiple comparisons. ****p < 0.0001, **p < 0.01, *p < 0.05.

We next transfected HEK cells with empty vectors or plasmids encoding human SAA1, collected and concentrated the conditioned media, and measured production of SAA1 of ∼30–40 ng/ml (Fig. 2A). However, when exposed to J774 cells, these same SAA1-rich culture supernatants were not able to stimulate TNF-α production, in contrast to that elicited by the addition of 10 ng/ml apoSAA to the conditioned media from empty vector–transfected cells (Fig. 2B). In addition to its induction of proinflammatory cytokine production, we (4) and others (38) have reported that stimulation of DCs with apoSAA elicits the production of cytokines capable of promoting Th17 differentiation. However, exposure of DCs to conditioned media from empty vector– or human SAA1–transfected HEK cells followed by subsequent polyclonal stimulation of naive CD4+ T cells in the DC-derived media was not able to decrease IFN-γ production or promote IL-4 and IL-17A production, in contrast to apoSAA (Fig. 2C–E). These results suggest that the proinflammatory and Th17-inducing effects of SAA may be limited to those proteins made in E. coli.

FIGURE 2.
FIGURE 2.

apoSAA, but not SAA1 produced from eukaryotic cells, induces TNF-α secretion from macrophages and promotes IL-17A production from CD4 T cells. HEK cells were transiently transfected with empty vector (pcDNA3) or plasmids encoding human SAA1 (hSAA1.pcDNA3 or hSAA1.pCMV6XL5). Forty-eight hours later, culture supernatants were collected and concentrated using Amicon Ultra centrifugal concentrators, SAA1 concentrations were measured by ELISA (A), and the culture supernatants were used to stimulate J774 cells for 24 h (in the absence or presence of 10 ng/ml apoSAA), after which TNF-α production was measured by ELISA (B). Similarly prepared HEK supernatants were added to bone marrow–derived DCs (BMDCs) for 48 h, their culture supernatants were collected and added to CD4+ T cells that were stimulated with 5 μg/ml immobilized anti-CD3 and 2 μg/ml soluble anti-CD28 for 72 h, after which IL-4 (C), IFN-γ (D), and IL-17A (E) were measured by ELISA. Data are mean ± SEM and are representative of three (A and B) or two (C–E) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett (A and B) or Tukey (C–E) test for multiple comparisons. *p < 0.05, ****p < 0.0001.

Human SAA1 proteins made in E. coli stimulate TLR2

We (4) and others (5–18) have previously reported that the innate immune-stimulating capacity of apoSAA is not due to contaminating endotoxin (LPS) stimulating TLR4 but is instead mediated by protein in the preparation through stimulation of TLR2. Interestingly, the TLR2-stimulating capacity of human SAA1 has been demonstrated almost exclusively through the use of E. coli–derived hSAA1 proteins either generated by the investigators (6) or purchased from a reputable commercial vendor (5–17). A relatively common posttranslational modification of bacterial proteins is through lipidation (acylation), which occurs at specific cysteine amino acids in the context of appropriate neighboring amino acids, a sequence defined as a lipobox (39). Heterodimers of TLR2 and TLR6 signal in response to diacylated lipoproteins, whereas heterodimers of TLR2 and TLR1 signal in response to triacylated lipoproteins. As apoSAA has been previously reported to signal through both TLR1 and 2 heterodimers (7), we transfected HEK cells with TLR1, TLR2, or both TLR1 and TLR2, then stimulated the cells with commercially sourced recombinant SAA proteins. E. coli–derived human SAA1 proteins were able to stimulate TLR2- or TLR1/2-transfected cells and induce the production of IL-8, whereas mouse SAA1 or human SAA1 made in eukaryotic (HEK) cells were not (Fig. 3). Because the human SAA1 and apoSAA protein sequences do not contain a lipobox or even a cysteine that is required for acylation, these results imply that bacterial lipoproteins may be present in the E. coli–derived SAA1 recombinant proteins and mediate the TLR2-dependent proinflammatory effects ascribed to SAA.

FIGURE 3.
FIGURE 3.

apoSAA stimulates proinflammatory cytokine production via TLR2. HEK cells were exposed to a transfection reagent (PEI) or were transiently transfected with plasmids encoding human TLR1, TLR2, or both TLR1 and TLR2 (TLR1/2). After 48 h, the cells were then unexposed (control) or stimulated with 1 μg/ml of SAA from different sources for 24 h, and IL-8 was measured by ELISA. Data are mean ± SEM and are representative of three independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Tukey test for multiple comparisons. ****p < 0.0001.

Lipoproteins associated with human SAA1 elicit proinflammatory cytokine production

To determine whether bacterial proteins were present in apoSAA, we separated the preparation by reducing SDS-PAGE, extracted proteins from 12 gel slices, performed trypsin digestion, and analyzed tryptic peptides by mass spectrometry. We easily visualized on the stained gel a predominant 12-kDa band in the apoSAA prep, as was expected based on its molecular mass (Fig. 4A). An identical pattern was observed by the more sensitive technique of silver staining (data not shown), indicating that there were no other proteins present besides SAA that were particularly abundant. Although the only human protein sequence present in our analysis was SAA1, the gel lane also contained fragments from 91 E. coli proteins, 15 of which were probable or predicted lipoproteins (Fig. 4B). Furthermore, mass spectrometry analysis of tryptic peptides from additional commercial sources of SAA1 showed that only human SAA1 proteins derived from E. coli contained bacterial lipoproteins, whereas mouse SAA1 made from E. coli, human SAA1 made in HEK cells, and apoA1 made from E. coli by the same manufacturer as the E. coli–derived apoSAA and hSAA1 did not contain E. coli lipoproteins (Fig. 4C, 4D). These data implicate the selectivity of human SAA1 for interacting with specific bacterial proteins and lipoproteins, and they imply the possibility that this activity is part of the protein’s proinflammatory function.

FIGURE 4.
FIGURE 4.

apoSAA contains bacterial lipoproteins. Fifty micrograms of apoSAA was reduced, denatured, and separated by PAGE (A). The 12 indicated regions were subjected to in-gel digestion with trypsin. The extracted tryptic peptides were analyzed by liquid chromatography–tandem mass spectrometry, and the resulting spectra were searched against a database for human and E. coli proteins, including predicted and probable E. coli lipoproteins (B). Six micrograms of SAA from different sources were digested in-solution and similarly analyzed by mass spectrometry. Total E. coli proteins identified (C) as well as predicted and probable E. coli lipoproteins identified (D) are indicated. Data are from two independent experiments.

We next sought to determine whether lipoproteins were necessary and sufficient for the proinflammatory effects of human SAA1. Therefore, we treated LPS, the triacylated lipopeptide Pam3CSK4, or apoSAA with LPL, which deactivates the capacity of bacterial proteins to stimulate TLR2 (40). Although LPL had no impact on LPS (Fig. 5A), LPL dose-dependently inhibited the capacity of both Pam3CSK4 (Fig. 5B) and apoSAA (Fig. 5C) to stimulate TNF-α production from J774 macrophages. We next exposed OVA or human SAA1 produced in HEK cells to Pam3CSK4, subjected the preparations to centrifugation through 3-kDa cutoff filters to remove the 1.5-kDa Pam3CSK4, and stimulated J774 cells with the unfiltered preparation or the retained (filtered) fractions that were readjusted to the original volume. Although neither human SAA1 nor OVA stimulated TNF-α production, and the unfiltered preparations containing Pam3CSK4 all stimulated TNF-α production, the filtered fractions from Pam3CSK4 incubated with human SAA1, but not from Pam3CSK4 incubated with OVA, stimulated TNF-α production (Fig. 5D). These results demonstrate that human SAA1 produced from eukaryotic cells can bind lipopeptides that stimulate proinflammatory cytokine production.

FIGURE 5.
FIGURE 5.

Lipopeptides associated with SAA confer its ability to stimulate TNF-α production. One hundred nanograms per milliliter LPS (A), 100 ng/ml Pam3CSK4 (Pam, [B]), and 1 μg/ml apoSAA (C) were incubated with LPL and then used to stimulate J774 cells. Culture supernatants were collected 24 h later, and TNF-α concentrations were measured by ELISA. Human SAA1 from OriGene produced in HEK cells (hSAA1) or OVA (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins >3 kDa using Amicon Ultra centrifugal concentrators (filtered). Control and filtered preparations were used to stimulate J774 cells for 24 h, after which TNF-α concentrations in culture media were measured by ELISA (D). Data are mean ± SEM and are representative of three (A–C) or two (D) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett test for multiple comparisons (A–C) or two-way ANOVA with Tukey test for multiple comparisons (D). ****p < 0.0001 compared with Pam (B), apoSAA (C), or control (D). ###p < 0.001 compared with unfiltered Pam + hSAA1 (D).

Discussion

Studies conducted using recombinant SAA made in E. coli have implicated this family of acute-phase proteins as proinflammatory mediators (5–18), yet several other reports demonstrate no proinflammatory effect of the endogenous protein enriched from human serum (19, 20) or in transgenic mice producing high levels of circulating human (20, 22) or mouse (21) SAA1. Our own previous work using recombinant apoSAA demonstrated that the proinflammatory activity of human SAA1 is mediated by TLR2 and is inhibitable by proteinase K, and is present in TLR4-deficient cells and not inhibitable by polymyxin B (4). Consequently, it is widely appreciated that the proinflammatory activities of SAA are not a consequence of endotoxin contamination. Instead, our studies demonstrate that bacterial proteins, including lipoproteins, associate with hSAA1 produced in E. coli and mediate the activation of TLR2 and Nlrp3 to induce the production of proinflammatory and Th17-promoting cytokines. This association between hSAA1 and bacterial lipoproteins appears to be necessary and sufficient for the cytokine-inducing effects, because even though recombinant apoA1 contained some bacterial proteins, they were not lipoproteins or known TLR2 agonists and were not able to induce TNF-α production from J774 macrophages. The ability of LPL to diminish apoSAA-induced TNF-α production is dose-dependent, significant, and substantial, but is incomplete. Although reasons for this are uncertain, a possibility includes inadequate access of lipoproteins to the enzyme’s active site, in contrast to the effective inactivation of the lipopeptide Pam3CSK4. Additionally, nonlipopeptides, such as OmpA, are present in the apoSAA preparation that remain capable of stimulating TLR2 and inducing TNF-α production despite LPL. Although LPL has activity on both lipoteichoic acids and lipoproteins (40), Gram-negative E. coli do not contain lipoteichoic acid, implying that the proinflammatory activity of hSAA1 is indeed largely conferred by a lipoprotein. In addition to their ability to stimulate TLR2, lipoproteins such as palmitate-conjugated albumin can serve as a strong signal for activation of the intracellular pattern recognition receptor, Nlrp3 (41). Although SAA is not directly palmitoylated, perhaps contaminating bacterial (lipo)proteins are also responsible for the Nlrp3-stimulating capacity of apoSAA (4, 11, 17, 36), especially considering the capacity of Pam3CSK4 to augment IL-1β release.

The acylation of bacterial lipoproteins occurs at the cell membrane (39), whereas recombinant proteins accumulate in intracellular inclusion bodies prior to acylation (42). Consequently, it is likely that the association of hSAA1 and bacterial lipoproteins occurs during processing to extract the recombinant protein, not during their biosynthesis, implying that similar associations may also occur between circulating SAA and bacterial proteins in vivo during infection. Interestingly, the E. coli–derived recombinant apoSAA and hSAA1 preparations used in our studies contained OmpA, a bacterial protein previously reported to activate macrophages (43) and DCs (44) via TLR2 and to interact with SAA (27). The E. coli–derived hSAA1 proteins also contained several bacterial lipoproteins, including periplasmic methionine binding lipoprotein (MetQ), peptidoglycan-associated lipoprotein (Pal), DUF3053 family lipoprotein (YiaF) and the lipoproteins DcrB and AcrA (Supplemental Table I). However, neither apoSAA nor hSAA1 contained the triacylated Braun Lipoprotein that is abundant in the E. coli outer membrane and stimulates TLR2 (45). Furthermore, none of these bacterial proteins were present in the E. coli–derived recombinant mouse SAA1 used in our studies.

Our results support several earlier studies implicating that acute-phase SAA lacks proinflammatory activity (19–21). Interestingly, these studies were conducted using SAA enriched from the serum/plasma of subjects with rheumatoid arthritis, a sterile inflammatory disease, or from cardiac surgery patients and associated with HDL. It will be important to examine whether during bacterial infection the highly induced levels of SAA, some of which exist as soluble proteins independent of HDL but can also form HDL-sized complexes with phospholipids (30), interact with bacterial lipoproteins and gain proinflammatory activity.

The reasons for the distinct differences between human and mouse SAA1 proteins to associate with bacterial (lipo)proteins are uncertain. Despite their evolutionary relatedness based on genomic organization and regulation, the secreted form of human SAA1 has 75% amino acid identity with mouse SAA1 [by Basic Local Alignment Search Tool (46)], which may account for the aforementioned differences. However, there is 80% identity between the N-terminal sequences of human SAA1 (aa 11–58) that have been reported to confer SAA’s TLR2-dependent proinflammatory effects (18) and those in mouse SAA1. Although human SAA1 derived from HEK cells did not promote the Th17 differentiation induced by E. coli–derived human SAA1, it is very interesting that SAA1/2 double knockouts elicit diminished responses to the Th17 induction elicited by Segmented Filamentous Bacteria infection, and that high concentrations of recombinant mouse SAA1 were able to increase IL-17A and IL-17F production in a DC-independent manner (47). These results suggest additional activities of SAA1 in innate and adaptive immunity that are perhaps related to cell survival and immunometabolism (48). In fact, endogenous SAA1 isolated from human serum that lacked proinflammatory effects did enhance the survival of primary human neutrophils by promoting antiapoptotic pathways (19). This ability of SAA1 to affect inflammatory cells may be due to differences between recombinant and endogenous forms of SAA1, differences between the responses of primary cells in the setting of acute inflammation in vivo, differential SAA isoform expression at sites of inflammation, and the stimulation of cells through receptors besides TLR2, TLR4, and NLRP3 (e.g., FPR2/FPRL1, SB-R1, and RAGE) that have been implied to elicit the effects of SAA.

Our results call for a re-evaluation of the proinflammatory effects ascribed to SAA through the use of E. coli–derived recombinant proteins. For researchers requiring a recombinant form of human SAA for experimentation, it is reassuring that a commercial source made in eukaryotic cells and containing no bacterial proteins is available. It is highly recommended that such a eukaryotic cell–derived source of SAA be used for all future studies in which its potential participation in inflammation is evaluated.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

This paper is in honor of Dr. Edward Burgess, a promising doctoral candidate whose life was cut short by malignant mesothelioma and who was posthumously conferred the Ph.D. degree. We thank Marion Weir for helping with some of the mass spectrometry analyses. We thank Dr. Renee Stapleton and Sara Ardren for assistance in procuring human blood samples.

Footnotes

  • This work was supported by National Institutes of Health Grants R01 HL107291, R01 HL133920 (both to M.E.P.), R01 AI103003 (to M.J.W.), T32 HL076122 (to E.J.B. and M.J.R.), P30 GM103532, P20 GM103496, and P20 GM103449. Research reported in this publication was supported by an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health under Grant P20 GM103449. The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    apoA1
    human apolipoprotein A1
    apoSAA
    the apolipoprotein form of human SAA1
    DC
    dendritic cell
    FPLC
    fast protein liquid chromatography
    HDL
    high-density lipoprotein
    HEK
    human embryonic kidney
    hSAA1
    recombinant human SAA1
    Kav
    elution volume parameter
    LPL
    lipoprotein lipase
    LSM
    lymphocyte separation medium
    OmpA
    outer membrane protein A
    PEI
    polyethylenimine
    SAA
    serum amyloid A
    Vo
    void volume.

  • Received April 11, 2018.
  • Accepted August 4, 2018.

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