Published July 3, 2017, doi:10.4049/jimmunol.1700470 The Journal of Immunology

Suppression of Lipopolysaccharide-Induced Inflammatory Response by Fragments from

Huibin Zhou,*,1 Mingjie Chen,*,1 Gufang Zhang,* and Richard D. Ye*,†

Serum amyloid A (SAA) is known as an acute-phase and a biomarker for inflammatory diseases. Published studies have shown that SAA possesses proinflammatory cytokine-like activity and is chemotactic for phagocytes, but the structural basis for these activities remains unidentified. In this article, we report that truncated SAA1 lacking N- and C-terminal sequences exhibit reduced proinflammatory activity and strongly suppress LPS-induced expression of IL-1b, IL-6, and TNF-a in macro- phages. A truncated SAA1 containing aa 11–58 was examined further and found to facilitate p38 MAPK phosphorylation while reducing LPS-stimulated phosphorylation of ERK and JNK. In LPS-challenged mice, aa 11–58 reduced the severity of acute lung injury, with significantly less neutrophil infiltration in the lungs and attenuated pulmonary expression of IL-1b, IL-6, and TNF-a. Coadministration of aa 11–58 markedly improved mouse survival in response to a lethal dose of LPS. A potent induction of IL-10 was observed in a TLR2-dependent, but TLR4-independent, manner in stimulated with aa 11–58. However, the aa 11–58 fragment of SAA1 was unable to induce chemotaxis or calcium flux through formyl peptide receptor 2. These results indicate that the N- and C-terminal sequences contain structural determinants for the proinflammatory and chemotactic activities of SAA1, and their removal switches SAA1 to an anti-inflammatory role. Given that proteolytic processing of SAA is associated with the pathological changes in several diseases, including secondary amyloidosis, our findings may shed light on the structure– function relationship of SAA1 with respect to its role in inflammation. The Journal of Immunology, 2017, 199: 000–000.

he acute-phase response (APR) is a systemic response to from constitutive apoptosis (10). Studies have also shown that SAA drastic changes, including trauma, infection, and surgery, stimulates expression of regulatory cytokines, such as T that may harm the host. APR is highly conserved in IL-33 (11), and exerts an effect on the epigenetic regulator Jmjd3 mammals and is characterized by the rapid rise in serum levels of (12). More recent studies have shown that acute-phase SAA pro- proteins termed the acute-phase reactants (1). Serum amyloid A teins are expressed in gut epithelial cells and play a role in pro- (SAA) and C-reactive protein are two major acute-phase proteins moting the Th17 response through the gut microbiome (13, 14). whose serum concentrations may increase by .1000-fold within The human SAA proteins are encoded by three : SAA1, 48–72 h after the initiation of APR (2). Numerous studies have SAA2, and SAA4; SAA1 and SAA2 are inducible, and SAA4 is shown that SAA and C-reactive protein are clinical biomarkers of expressed constitutively (15). Another member of the SAA inflammatory diseases, such as rheumatoid arthritis (3, 4), al- family, SAA3, is a pseudogene in humans but encodes a functional though whether and how these proteins actually play a role in protein in mice (16). Among these SAA proteins, SAA1 has been inflammation were unclear for many years. In vitro studies have the most extensively investigated for its functions in binding high- shown that recombinant SAA is chemotactic for phagocytes (5) density lipoprotein and regulation of lipid metabolism (17). Ma- and is able to induce the expression of inflammatory cytokines and ture SAA1 contains 104 aa and is present in a four-helix bundled chemokines, including IL-1b, TNF-a, IL-6, and IL-8 (6–8). When structure, based on a recently solved crystal structure (18). No injected into mice, recombinant SAA proved to be a potent in- conserved functional domains or motifs have been identified in the ducer of G-CSF, which leads to neutrophilia (9). Exposure of SAA structure. As a result, little is known about the structure– neutrophils to SAA rescues these terminally differentiated cells function relationship of mature SAA. Amyloid A is a cleavage product of SAA that forms fibrils in the lesions of secondary amyloidosis (19). Pathological characterization of secondary am- *Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Edu- yloidosis found amyloid fibrils with extended b-sheets. However, cation, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; and †Institute of Chinese Medical Sciences, University of Macau, Macau no extended b-sheets were found in the crystal structure of SAA1 Special Administrative Region 999078, China (18). Evidence suggesting the complexity of the SAA structure 1H.Z. and M.C. contributed equally to this work. also comes from published reports that SAA is a potent agonist for Received for publication March 30, 2017. Accepted for publication June 6, 2017. several cell surface receptors, including the formyl peptide re- This work was supported by Grant 31470865 from the National Natural Science ceptor 2 (FPR2) (20), TLR2 (21), TLR4 (22), the receptor for Foundation of China. R.D.Y. received support from the Science and Technology advanced glycation end products (23), and P2X7 (24). Because Development Fund of Macau (Grant 026/2016/A1) and the University of Macau (Grants CPG2015-00018-ICMS and SRG2015-00047-ICMS-QRCM). these receptors have different structures, it is predicted that SAA uses multiple interfaces for its interaction with the ectodomains Address correspondence and reprint requests to Dr. Richard D. Ye, University of Macau, Avenida da Universidade, Building N22, Room 1049, Macau 999078, China. of the receptors. In an attempt to delineate the structure–func- E-mail address: [email protected] tion relationship of SAA1 in immune cells, we dissected SAA1 The online version of this article contains supplemental material. by removing its N- and C-terminal amino acids and testing these Abbreviations used in this article: APR, acute-phase response; BMDM, bone marrow– SAA1-derived fragments on macrophages and in an LPS- derived macrophage; FPR2, formyl peptide receptor 2; MPO, myeloperoxidase; SAA, induced sepsis model. Our results show that amino acids at serum amyloid A; WT, wild-type. the N and C termini contain important structural determinants Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 for the proinflammatory and chemotactic properties of SAA1,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700470 2 SAA FRAGMENTS SUPPRESS LPS RESPONSE because the truncated forms of SAA1 exhibit strong anti-inflammatory before (27), using recombinant SAA1 or the aa 11–58 fragment of SAA1 activities. as agonist. Statistical analysis Materials and Methods Reagents Statistical significance among different samples was assessed with a paired Student t test or one-way ANOVA with repeated measures if comparing LPS from Escherichia coli (serotype 0111:B4) was obtained from Sigma- more than two treatment groups. Statistical significance was defined as Aldrich (St. Louis, MO). The Abs against phosphorylated p38 (4631S), *p , 0.05 and **p , 0.01. Analysis and graphing were performed using ERK (4370S), and JNK (4668S) were from Cell Signaling Technology Prism 5.0 software (GraphPad, San Diego, CA). (Danvers, MA). Donkey anti-rabbit IRDye 800CW–conjugated secondary Ab was from LI-COR (Lincoln, NE). Results Mice Removal of N- and C-terminal amino acids altered the cytokine-like activities of SAA Male C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). Tlr2-knockout mice were purchased from The Based on recently resolved structures of SAA1 (18), its monomer Jackson Laboratory (Bar Harbor, ME). C57BL/10ScN (Tlr4lps-del) mice contains a four-helix bundle structure with an N-terminal sequence were obtained from the Nanjing University Model Animal Research In- showing an amyloidogenic property and a C-terminal fragment (aa stitute (Nanjing, China). Experiments involving mice were performed in a accordance with the Statute on the Administration of Laboratory Animals 89–104) interacting with several residues on the helices for its by the Ministry of Science and Technology of China. All animal studies stabilization (18). Therefore, the SAA1 monomer and its native were conducted at Shanghai Jiao Tong University with age- and sex- hexamer possess numerous surface structures to interact with matched littermates, using procedures approved by the Institutional Ani- multiple proteins, including cell surface receptors in immune cells mal Care and Use Committee of Shanghai Jiao Tong University. For survival and acute lung injury assays, 6–8-wk-old male C57BL/6 (8). To identify the structural determinants responsible for the mice were given i.p. injections of LPS or LPS plus SAA1-derived peptides. cytokine-like activities of SAA1, we prepared deletion mutants of Mice (10 in each group) were observed for 72 h for survival. For acute lung SAA1 by removing amino acids from the N and C termini injury analysis, the treated mice were sacrificed after 24 h, and lung tissues (Fig. 1A). The study was designed with the prediction that removal were collected. of seven or more amino acids from the N terminus might result in Protein expression SAA1 fragments that were no longer amyloidogenic, whereas re- moval of amino acids from the C terminus might relax the helix- SAA1-derived fragments were expressed in the yeast species Pichia pastoris. Briefly, the corresponding DNA fragments were obtained using PCR and bundle structure of the SAA1 monomer. The truncated SAA1 the human SAA1.5 cDNA as template. The DNA fragments were cloned proteins were expressed in the yeast P. pastoris to minimize LPS into the pPIC9K plasmid vector (Invitrogen, Carlsbad, CA) downstream of contamination (see Materials and Methods) (Fig. 1B). Although a His6-SUMO tag and a tobacco etch virus protease cleavage site. The ex- SAA1 fragments of various lengths were designed, only those that pression conditions were those described by the manufacturer. The products werepurifiedbyNi2+ affinity chromatography and cleaved by tobacco etch were readily expressed in the recombinant protein expression sys- virus protease to remove the His6-SUMO tag. The expression and purification tem (Fig. 1A) were tested. Analysis of the expressed fragments by of full-length SAA1 were described previously (25). size-exclusion chromatography found that they were present as monomers, whereas the full-length SAA1 appeared to be multi- Cytokine expression merized (see Supplemental Fig. 1 for example). Bone marrow–derived macrophages (BMDMs) were prepared from wild- Previous in vitro studies showed that macrophages respond to type (WT) and knockout mice, as described previously (26). Culture recombinant SAA with elevated expression of proinflammatory medium of BMDMs treated with LPS or SAA1-derived peptides was a b collected at the time points indicated in the figure legends. The cells were cytokines, such as TNF- , IL-1 , and IL-6 (reviewed in Ref. 8). collected for total RNA preparation and real-time PCR analysis of cytokine To determine whether the reported cytokine-like activities of SAA transcripts. could be attributed to any structures of the mature protein, we MAPK phosphorylation stimulated mouse BMDMs with the truncated SAA1 proteins and measured the transcripts of IL-6, TNF-a, and IL-1b. As shown in A stable line of HeLa cells expressing human TLR2 (21) was used in Fig. 1C, the truncated SAA1 proteins exhibited significantly re- MAPK-phosphorylation assays. The experimental conditions were identi- cal as described previously (21), with the exception that SAA1 and the aa duced capabilities to induce these proinflammatory cytokines. 11–58 fragment were used as agonists. The cells were stimulated for Macrophages respond to LPS with robust expression of proin- various times, as indicated, or in the presence of LPS, and phosphorylated flammatory cytokines. We determined whether the truncated SAA1 MAPKs were detected with Western blotting using Abs against phos- proteins could alter the LPS-induced expression of proinflammatory phorylated ERK, JNK, and p38 MAPK. cytokines. When the truncated SAA1 proteins were added together Chemotaxis with LPS to BMDM culture, significantly reduced cytokine ex- pression was observed (Fig. 1D). SAA1 fragments aa 11–68 and aa A stable line of rat basophilic leukemia (RBL-2H3, CRL 2256; American Type Culture Collection, Manassas, VA) expressing human FPR2 (FPR2- 11–58 were particularly potent in blocking the cytokine-induction RBL) (27) was used in a chemotaxis assay, carried out in a 48-well effect of LPS. The aa 11–58 fragment was analyzed in more detail microchemotaxis chamber (Neuro Probe, Cabin John, MD) through a at different concentrations. As shown in Fig. 1E, it dose depen- m polycarbonate filter of 10 m pore size. The procedure was described in dently attenuated the LPS-induced expression of the transcripts of detail in a previous publication (28), and had an incubation time of 4 h. a b Chemotaxis index was calculated as the number of cells migrating toward IL-6, TNF- , and IL-1 at the mRNA level. stimuli/the number of cells migrating toward medium. Checkerboard Fragment aa 11–58 protected mice against LPS-induced analysis was conducted to verify that migration of the cells occurred only when a higher concentration of the chemoattractant was present in the inflammation and death lower wells of the chamber. Male C57BL/6 mice (6–8 wk, 10 per group) were given LPS Calcium mobilization (25 mg/kg body weight) or PBS as a negative control. In the test group, the aa 11–58 fragment (5 mg/kg body weight) was mixed with FPR2-RBLs were incubated with the calcium indicator Calcium 5 reagent, according to the manufacturer’s instruction (Molecular Devices, Sunny- LPS before peritoneal administration. The mice were then observed vale, CA). Calcium mobilization was measured on a FlexStation 3 Multi- for 72 h, and the survival rate was calculated. As shown in Fig. 2A, Mode Microplate Reader (Molecular Devices) essentially as described all mice receiving PBS survived, whereas 90% of the mice receiving The Journal of Immunology 3

FIGURE 1. Preparation and initial characterization of SAA1-derived frag- ments. (A) Schematic representation of humanSAA1initsmatureformand the location of the truncated SAA1 proteins named after their starting and ending amino acid positions. Filled bars of the full-length SAA1 depict a-heli- ces. (B) Recombinant SAA1 fragments with expected sizes separated by tricine SDS-PAGE. A representative gel im- age from three experiments, all pro- ducing similar results, is shown. (C) Relative mRNA levels of IL-6, TNF-a, and IL-1b in mouse BMDMs stimulated for 8 h with SAA1 or the fragments (0.5 mM each). Data are mean 6 SEM based on three inde- pendent experiments. *p , 0.05, **p , 0.01 versus SAA1 stimulation alone. (D)Effectofthefragments(0.5mM) on LPS (100 ng/ml)-induced expression of IL-6, TNF-a, and IL-1b,shownas relative mRNA levels of these cyto- kines in the absence or presence of the SAA1 fragments. Data are mean 6 SEM based on three independent experiments. *p , 0.05, **p , 0.01 versus LPS stimulation only. (E) BMDMs were stimulated for 4 h by LPS (100 ng/ml), with or without the aa 11–58 fragment at the indicated con- centrations. The relative mRNA levels of IL-6, TNF-a,andIL-1b were quan- tified. Data are mean 6 SEM based on three independent experiments. Tripli- cate measurements were taken in (C–E). *p , 0.05, **p , 0.01 versus LPS only.

LPS died after 48 h. In comparison, 60% of the mice that received able to stimulate the phosphorylation of ERK to a similar extent in LPS + fragment aa 11–58 survived through the experiment. TLR2-HeLa cells, the aa 11–58 fragment was not able to stimulate In a parallel experiment, the mice receiving the above treatments JNK phosphorylation. The aa 11–58 fragment was more potent were sacrificed after 24 h for analysis of lung structure, cytokine than the full-length SAA1 in the induction of p38 MAPK phos- expression, and myeloperoxidase (MPO) activity, which re- phorylation. Phosphorylation of p38 MAPK appeared as early as flects the number of infiltrating neutrophils (Fig. 2B–D). The 2 min after aa 11–58 stimulation, but it was not detectable in HeLa LPS-induced increase in lung MPO activity was attenuated sig- cells without stable expression of TLR2 (Supplemental Fig. 2). nificantly in the presence of the aa 11–58 fragment, whereas ad- Altered receptor selectivity may be attributable to the difference ministration of the fragment alone did not alter MPO activity between the aa 11–58 fragment and the full-length SAA1 in (Fig. 2B). The increase in IL-6, TNF-a, and IL-1b expression in stimulating MAPK phosphorylation, as discussed below. LPS-stimulated macrophages was reduced significantly when LPS is a potent activator of major forms of MAPKs, including ERK, fragment aa 11–58 was present. An examination of lung tissue p38, and JNK. In BMDMs, costimulation with LPS and the aa 11–58 sections found fewer infiltrating leukocytes in the LPS + aa 11–58 fragment reduced the phosphorylation of ERK and JNK compared group (Fig. 2D, lower right panel) compared with the group given with LPS stimulation alone. An enhanced phosphorylation of p38 LPS alone (Fig. 2D, upper right panel). Taken together, these MAPK was also observed (Fig. 4). These findings suggest that the aa results show that the truncated SAA1 protects mice against LPS- 11–58 fragment differentially regulates MAPK activation. induced acute lung injury. Fragment aa 11–58 potently induced IL-10 expression in a Fragment aa 11–58 differentially regulated MAPK activation TLR2-dependent manner In a previous study, SAA was shown to stimulate the phosphor- One of the downstream effectors of p38 MAPK is IL-10, a major ylation of ERK, p38 MAPK, and JNK through TLR2 (21). This anti-inflammatory cytokine (29). Although the aa 11–58 fragment activity was altered when the truncated SAA1 was used (Fig. 3). lacked the ability to induce the proinflammatory cytokines IL-6, Although the full-length SAA1 and the aa 11–58 fragment were TNF-a, and IL-1b, it induced a potent expression of IL-10 at the 4 SAA FRAGMENTS SUPPRESS LPS RESPONSE

FIGURE 2. Effect of the aa 11–58 fragment on mouse survival and acute lung injury following LPS challenge. (A) C57BL/6 mice (10 in each group) were given a peritoneal injection of PBS, LPS (25 mg/kg body weight), or LPS plus the aa 11–58 fragment (5 mg/kg body weight). The survival of mice was followed for 72 h. (B–D) In parallel experiments, the treated mice were sacrificed at 24 h, and lung tissues were collected. (B) MPO activity in the lung tissue was compared. (C) The relative levels of mRNA for IL-6, TNF-a, and IL-1b were determined in the lung tis- sues. Data in (B) and (C) are mean 6 SEM based on three independent experiments, each in triplicates. (D) Sections of lung tissue from the different treatment groups were stained with H&E. A set of representative images is shown. *p , 0.05, **p , 0.01.

mRNA and protein levels (Fig. 5A, 5B). In BMDMs, maximal and plays a role in the tumor microenvironment (22, 30). We IL-10 mRNA expression was measured at 4 h, and the IL-10 compared BMDMs from Tlr22/2 and Tlr42/2 mice for their protein level peaked at 8 h after stimulation. In comparison, abilities to respond to SAA1 and the aa 11–58 fragment with LPS was a weak inducer of IL-10 expression (Fig. 5C, 5D). When IL-10 production. As shown in Fig. 5E–H, the aa 11–58 fragment used together, LPS did not significantly alter the IL-10–inducing was several times more potent than the full-length SAA1 in the activity of the aa 11–58 fragment. induction of IL-10 expression at the mRNA and protein levels. TLR2 is one of the functional receptors of SAA (21). TLR2 The absence of Tlr2 markedly attenuated the induced expression mediates a number of known activities of SAA in vitro and of IL-10, contributing to an ∼77% reduction in IL-10 expression in vivo, including the ability to induce the expression of IL-12p40, in BMDMs stimulated with the aa 11–58 fragment and an ∼73% IL-23p19, and G-CSF (9, 21). TLR4 is another SAA receptor that reduction in cells stimulated with full-length SAA1 (Fig. 5E, mediates the SAA-induced expression of inducible NO synthase 5F). In comparison, deletion of Tlr4 did not significantly alter The Journal of Immunology 5

FIGURE 3. Comparison of the aa 11–58 fragment with SAA1 for MAPK phosphor- ylation. (A) TLR2-HeLa cells were stimu- lated for the indicated time with SAA1 (left panel) or the aa 11–58 fragment of SAA1 (right panel). The samples were resolved on SDS-PAGE, blotted, and detected with Abs against the phosphorylated forms of ERK, p38 MAPK, or JNK. (B) The relative phos- phorylation level (phospho-MAPK/total MAPK) was quantified and shown with time 0 set as 1, against which other samples were compared. Data shown are mean 6 SEM based on three independent experiments. *p , 0.05, **p , 0.01 versus samples stimulated with SAA1 in the same time groups.

the induction of IL-10 by full-length SAA1 or by the aa 11–58 C-terminal sequence are typically found in the lesions of sec- fragment (Fig. 5G, 5H). These findings suggest that TLR2 is ondary amyloidosis, in the form of amyloid A aggregates (34, 35). primarily responsible for the induced expression of IL-10 by the These fragments are degradation products of SAA that are aa 11–58 fragment, as well as by full-length SAA1. missing between 15 and 83 aa from the C terminus (19). In addition to the two TLRs, FPR2 binding of SAA1 contributes Proteinases found in inflammatory tissues, including matrix to its proinflammatory activities through induced cell migration, metalloproteinases, are known to degrade SAA into fragments of release of the chemokine IL-8, and tissue-damaging granular en- various lengths (19, 36, 37). A more recent study of SAA1 and zymes (20, 31). To determine whether FPR2-interacting capability activation of the NALP3 proteosome suggests that secreted ca- is retained in the truncated SAA1 proteins, the aa 11–58 fragment thepsin B may contribute to extracellular processing of SAA (38). was compared with full-length SAA1 in chemotaxis and calcium Therefore, although the tested SAA1 fragments, including aa 11– flux assays using FPR2-RBLs (27). As shown in Fig. 5I–K, the aa 58, have not been found in vivo, SAA1 fragments of similar sizes 11–58 fragment showed no chemotactic activity at FPR2 and was and compositions may be generated in vivo by these proteases; the unable to induce calcium mobilization through this chemo- present study may help to gain an understanding of the structure– attractant receptor. function relationship of SAA1 with respect to its role in the reg- ulation of inflammation. Because of technical limitations, many of Discussion the designed SAA1 fragments were not expressed efficiently in The bioactivity of SAA has drawn increasing attention because of our recombinant protein–expression system. As a result, we were recent reports on its immunomodulatory functions in mice (13, 14). unable to determine the minimal size and the composition of the Data from in vitro and ex vivo studies suggest that SAA possesses fragments that retain the observed functional properties. Future potent activities in the upregulation of inflammatory cytokines (8). studies may use synthetic peptides instead of recombinant protein However, the structural basis of such activities remains largely expression. unknown. The results from this study demonstrate for the first We previously reported that full-length SAA could induce a time, to our knowledge, that the N- and C-terminal residues of modest level of IL-10 expression, along with potent expression of a SAA1 are required for its chemotactic activity at FPR2 and for plethora of proinflammatory cytokines (21). The finding that the aa optimal induction of proinflammatory cytokines, such as IL-6, 11–58 fragment potently induces IL-10, but not major proin- TNF-a, and IL-1b. Moreover, removal of amino acids from the flammatory cytokines, is of interest. IL-10 is an anti-inflammatory N and C termini diminishes the proinflammatory activity of cytokine that plays an important role in the resolution of inflam- SAA1, as well as bestows anti-inflammatory activities upon the mation and in tissue repair (29). As a result, the induced expres- resulting fragments, contributing to an enhanced induction of sion of IL-10 may attenuate the inflammatory response, thereby IL-10 and reduced response to LPS. It was previously reported reducing acute lung injury and improving the survival of mice, as that the N-terminal 7–10 aa of SAA1 constitute an amyloidogenic seen in our experiments. It is unclear whether the increased IL-10 determinant (19, 32). Removal of these amino acids, as seen with secretion is entirely responsible for the anti-inflammatory phe- cathepsin D treatment (33), generates proteolytic fragments that notype in mice receiving the aa 11–58 fragment. Because the anti- are less likely to form amyloid fibrils. Indeed, there was no sign of inflammatory activities were observed in experiments using LPS, aggregation of the aa 11–58 and other truncated SAA1 proteins it is possible that the SAA1 fragment binds to LPS and impedes its shown in Fig. 1A during their preparation. Size-exclusion chro- interaction with TLR4. Given that SAA1 is a known ligand for matography showed that the aa 11–58 fragment remained a TLR4, it is also possible that the SAA1 fragment may target TLR4 monomer, whereas full-length SAA1 appeared as a multimer and block LPS binding to its cell surface receptor. However, (Supplemental Fig. 1). The difference may contribute to their coadministration of LPS with the aa 11–58 fragment did not varied properties with regard to receptor selectivity and affinity, as significantly alter the IL-10–inducing property of aa 11–58 evidenced in some of the functional assays, including MAPK (Fig. 5C, 5D), suggesting that a direct interaction between the phosphorylation and chemotaxis. SAA fragments with a reduced two is less likely or it does not abrogate the IL-10–inducing 6 SAA FRAGMENTS SUPPRESS LPS RESPONSE

FIGURE 4. Differential regulation of MAPK phosphorylation by the aa 11–58 fragment. (A) Representative Western blots showing effects of the aa 11–58 fragment on MAPK phos- phorylation (lane 2) and on LPS-in- duced MAPK phosphorylation (lanes 4–6). BMDMs were stimulated with LPS (100 ng/ml) for 60 min, in the absence or presence of the aa 11–58 fragment at the indicated concentra- tions. The phosphorylation of MAPKs was detected using Abs against the phosphorylated forms of these kinases. Total MAPKs were also detected for comparison. (B) Quantification of the blots in (A) and from two similar ex- periments. *p , 0.05, **p , 0.01 versus samples stimulated with LPS alone.

effect of the fragment. Moreover, the aa 11–58 fragment does protein but may be altered with removal of its N- and C-terminal not seem to activate TLR4 directly for IL-10 induction sequences and the resulting change from multimers to monomers. (Fig. 5G, 5H). A study has shown that SAA3-derived peptides The present study uses fragments from human SAA1, which is may interact with MD-2, leading to MyD88-dependent acti- highly similar to mouse Saa1 in its primary sequence (76% iden- vation of TLR4 (39). Because the net results of these actions tical overall and 77% identical in the aa 11–58 region, based on are enhanced NF-kBactivationandincreasedIL-6andTNF-a mouse Saa1.1). Therefore, it is less likely that species differences in expression, the phenotypical changes induced by the SAA3 the primary sequence contribute to the different properties of the aa fragments are different from those caused by the aa 11–58 11–58 fragment. The diversity of the SAA-induced functions, ob- fragment. Based on these analyses, enhanced IL-10 production served in studies using human and mouse SAA, illustrates the remains a major contributing factor to the observed anti- complex interactions between SAA and cell surface receptors on inflammatory phenotype in macrophages and in mice treated different types of cells. It is postulated that the activities of SAA with the aa 11–58 fragment of SAA1, but other possibilities observed in vivo and in isolated primary cells result from activation have to be considered as well. The observation that aa 11–58 of multiple receptors in combinations. Evidence supporting this could induce p38 MAPK phosphorylation with a different ki- notion comes from several published studies. A comparison of netics than SAA1 suggests that the fragment may activate a SAA-induced cytokine profiles from WT and Tlr2-deficient mac- different set of receptors than full-length SAA1, most probably rophages identified major differences (21). Although the induced fewer receptors. As shown in Supplemental Fig. 2, untrans- expression of IL-23p19 and IL-1rn was largely dependent on TLR2, fected HeLa cells did not respond to the aa 11–58 fragment the induced expression of IL-18 was not altered at all in the absence with enhanced p38 MAPK phosphorylation, supporting the of Tlr2. In the current study, the aa 11–58 fragment seems to have notion that this response is TLR2 dependent. Therefore, it is retained its ability to activate TLR2, because it can stimulate IL-10 possible that the aa 11–58 fragment displays increased avidity expression in a TLR2-dependent manner. However, its ability to for some of the receptors (e.g., TLR2) but reduced avidity for induce proinflammatory cytokine expression is markedly dimin- other receptors (e.g., FPR2), as evidenced by the lack of che- ished. The seemingly perplexing observation suggests that optimal motactic activity. induction of proinflammatory cytokines, such as IL-1b, IL-6, and Because early studies of the inflammatory activities of SAAwere TNF-a, may require the activation of more than one receptor, conducted almost exclusively with the use of recombinant SAA1, whereas the induced expression of IL-10 by SAA1 relies primarily which contains 2 aa substitutions from SAA2, there was suspicion on TLR2. Evidence supporting this explanation comes from a study that the observed cytokine-inducing activities of SAA might be an showing that optimal IL-1b production requires SAA activation of artifact caused by the recombinant protein (40, 41). With the use of TLR2, TLR4, and the P2X7 receptor in an NLRP3 inflammasome– SAA1/2-knockout mice (13, 14, 42) and transgenic expression of dependent manner (38). As the truncated SAA1 proteins lose their SAA1 (43), the cytokine-inducing capability of SAA has been abilities to simultaneously activate multiple SAA receptors, the func- confirmed in vivo. The present study provides additional evidence tional roles for the individual SAA receptors and their downstream that the cytokine-regulating activity is intrinsic to the SAA1 signaling pathways become more obvious. One of the signaling The Journal of Immunology 7

FIGURE 5. Induction of IL-10 expression by the aa 11–58 fragment is TLR dependent. BMDMs were stimulated with the aa 11–58 fragment for different time periods, and IL-10 mRNA (A) and protein (B) were measured by real-time PCR and ELISA, respectively. **p , 0.01 versus unstimulated samples (time 0). IL-10 mRNA (C) and protein (D) were determined in BMDMs stimulated with LPS (100 ng/ml), in the absence or presence of the aa 11– 58 fragment (0.5 mM), or stimulated with aa 11–58 alone. **p , 0.01 versus LPS alone. The aa 11–58 fragment and full-length SAA1 were compared for their ability to induce IL-10 mRNA (E) and protein (F) release in BMDMs from WT and tlr22/2 mice. Full-length SAA1 and the aa 11–58 fragment were used at the same concentration (0.5 mM). *p , 0.05, **p , 0.01, Tlr22/2 macrophages versus WT macrophages. (G and H) Same as in (E) and (F), with the exception that tlr42/2 BMDMs were compared with WT BMDMs. All data were based on three independent experiments with triplicate measurements. (I) Chemotaxis of FPR2-RBLs showing the difference between the aa 11–58 fragment and SAA1. *p , 0.05, **p , 0.01 versus SAA1 in the same-dose group. (J and K)Ca2+ mobilization measured in FPR2-RBLs stimulated with full-length SAA1 or the aa 11–58 fragment. The data in (J) represent one of the three similar experiments. Quantification of relative Ca2+ mobilization was based on three separate measurements. molecules downstream of TLR2 is p38 MAPK. Activation of p38 tation of the data presented in this article, because LPS is the only MAPK is known to lead to IL-10 expression (44). Therefore, the stimulant used in our experiments. An inflammatory response can altered preference of SAA1-induced phosphorylation of MAPKs in be evoked by many agents, ranging from infectious to noninfec- the case of the aa 11–58 fragment may be one of the contributing tious ligands, and it is still unclear whether the SAA1-derived factors for the switch from a proinflammatory phenotype to an anti- fragments can inhibit the inflammatory response induced by inflammatory phenotype. agents other than LPS. These important limitations call for addi- What is the physiological relevance of SAA1 fragments with tional investigations into the mechanisms by which the SAA1 anti-inflammatory activity, considering that the full-length SAA1 fragments exert anti-inflammatory actions. Despite these limita- exhibits strong proinflammatory activities? A possible scenario is tions, the approach presented in this article provides a useful tool that, in the early phase of acute inflammation, SAA1 and other for studies of the structure–function relationship of SAA1 with acute-phase SAA proteins attract neutrophils and other phagocytes respect to its immunomodulatory functions. Moreover, an SAA to the site of inflammation through their proinflammatory and fragment lacking a proinflammatory cytokine-inducing capability, chemotactic activities. As more proteases are building up locally, while gaining the ability to induce IL-10 expression, may be of the accumulated SAA protein may be digested to produce frag- potential value for the treatment of inflammatory diseases. The ments that help to resolve inflammation and promote tissue repair. potent inhibitory effect of the aa 11–58 fragment in LPS-induced A recently published study showed that SAA could induce se- acute lung injury also suggests potential therapeutic usage of the cretion of active cathepsin B, which may convert SAA protein into fragment in diseases caused by Gram-negative bacterial infection. digested fragments through extracellular processing (38). In this Further investigations will be required to examine the pharma- regard, SAA1 serves as a homeostatic factor in the regulation of cokinetic properties of the SAA1 fragments, as well as the detailed inflammation. Although this hypothesis may be tested further in mechanisms by which these fragments exert anti-inflammatory future experiments, caution should be exercised in the interpre- functions. 8 SAA FRAGMENTS SUPPRESS LPS RESPONSE

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