Published online before print November 8, 2007
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,
,
Departments of
* Molecular Sciences and
Ophthalmology, University of Tennessee Health Science Center,
The VA Medical Center, and
The Mid South Center for Biodefense and Security, Memphis, Tennessee, USA
1 Correspondence: Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA. E-mail: fre{at}utmem.edu
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Key Words: inflammation • bacterial • cytokines • T cells
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study of the immunomodulatory activity of microbial products is often complicated by the technical difficulties encountered in their purification. Harsh conditions that allow obtaining pure products may destroy their biological activities, while bland conditions that preserve them often yield preparations that are contaminated with other microbial products, each one with its own biological properties. This has repeatedly led to conflicting results and egregious misattribution of biological activities.
Gram-positive bacteria infections, the most prevalent bacterial infections in humans, cause a powerful inflammatory response, primarily triggered by bacterial cell wall components and in some pathogenic species, by secreted bacterial toxins.
The cell wall of Gram-positive bacteria consists of a thick layer of peptidoglycan (PGN), a polymer of alternating N-acetylglucosamine and N-acetylmuramic acid cross-linked by short peptides and teichoic acid. Spanning and protruding from this carbohydrate network are a variety of molecules including lipoproteins and lipoteichoic acid (LTA).
Numerous studies have demonstrated the ability of PGN and LTA to induce a proinflammatory response. PGN and LTA have been reported to act as TLR2 agonists [3
4
5
6
], although for both bacterial products, contradictory results have been published. In the case of PGN, recent studies [7
, 8
] have tested commercially available preparation of Staphylococcus aureus PGN (PG-Sa) or highly purified PGN obtained from different bacteria and came to the conclusion that the reported TLR2 agonist activity of PGN is a result of LTA or lipoprotein contamination. Although TLR2 may not be involved in the recognition of PGN, other PRR have been identified that can mediate an innate immune response to PGN. It has been demonstrated that Nod2 [9
, 10
]; neuronal apoptosis inhibitory protein, MHC class II transcription activator, incompatibility locus protein from Podospora anserina, and telomerase-associated protein leucine-rich repeat protein 3 (Nalp3) [11
]; and Nalp1 [12
], which belong to the NLR family of PRR, can sense the presence in the cytoplasm of muramyldipeptide (MDP), a PGN substructure, leading to NF-
B activation (Nod2) or inflammasome activation (Nalp1, Nalp3).
In addition to PGN and LTA, Gram-positive bacteria, such as S. aureus and Streptococcus pyogenes, possess a large array of toxins that severely affect the host immune response. Among these virulence factors are superantigens (SAg), a group of secreted proteins with the unique ability to bypass conventional antigen recognition by directly cross-linking MHC class II molecules on APC with the TCR (reviewed in ref. [13 ]). SAg contact the MHC molecule outside the antigenic peptide groove, are active in the nanomolar range, and demonstrate individual preferences for binding to TCR bearing a particular Vβ element. This leads to activation of a large proportion of resting T cells (up to 20% of the circulating T cells) and massive production of proinflammatory cytokines that further exacerbate the inflammatory response initiated by the cell wall components and can cause severe pathology, such as food poisoning, toxic shock syndrome, and scarlet fever. SAg have been identified in Gram-positive, Gram-negative, and mycobacterial species and are commonly encoded by mobile genetic elements.
In the course of studies aimed at characterizing the proinflammatory activity of PGN, we have discovered that commercially available preparations of Staphylococcal and Streptococcal PGN are contaminated by SAg that activate T cells, leading to production of T cell-specific cytokines, such as IL-17, and expansion of T cell populations expressing selected TCR Vβ elements.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant SAg (rSAg) Streptococcal pyrogenic exotoxin C (SpeC) and Streptococcal mitogenic exotoxin Z (SmeZ) were expressed in E. coli and purified as described previously [14 ]. Staphylococcal toxic shock syndrome toxin-1 (TSST-1) was purchased from Toxin Technologies (Sarasota, FL, USA).
Cell isolation
Human PBMC were isolated from Leukopacks by Ficoll-Hystopaque density gradient centrifugation. Monocytes and T cells were sequentially purified from human PBMC using MACS CD14 microbeads first and then CD4 microbeads (Miltenyi Biotech, Auburn, CA, USA). Purity was checked by staining with FITC-conjugated anti-CD14 and PE-conjugated anti-CD4 antibodies (Sigma Chemical Co., St. Louis, MO, USA) and FACScan analysis and found routinely to be higher than 96%.
RNase protection assay
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNase protection assay was performed using 4 µg total RNA using the Riboquant kit (BD PharMingen, San Diego, CA, USA), as described previously [15
]. The hCK-1 and hCK-5 multiprobe template sets were used. For the IL-17 template set, fragments of predetermined size for each transcript were obtained by RT-PCR from LPS-PMA-stimulated PBMC total RNA and were cloned (in the antisense orientation) in the HindIII and XhoI site of pcDNA3. The resulting plasmids were linearized by XhoI restriction and used, as a mixture, in a standard in vitro transcription reaction using T7 RNA polymerase. The size of each fragment is as follows: IL-17A, 368 bp; IL-17F, 317 bp; IL-23 p19, 270 bp; IFN-
, 200 bp; IFN-inducible protein 10 (IP-10), 180 bp; GAPDH, 150 bp. The sequence of the primers used for the amplification is available upon request.
Cytokine measurements
IL-17 levels in conditioned supernatants were measured by ELISA using the paired antibodies kit from eBioscience (San Diego, CA, USA).
Luciferase assay
The HeLa-TLR2 cell line [15
] was transiently transfected in 24-well plates using Effectene reagent (Qiagen, Valencia, CA, USA) with 0.4 µg endothelial leukocyte adhesion molecule (ELAM)-luciferase and 0.2 µg pcDNA-CD14 and 0.1 µg CMV-β-galactosidease (β-Gal). Forty-eight hours after transfection, cells were stimulated for 6 h with different agonists. Luciferase assay was performed using Promega (Madison, WI, USA) reagents, according to the manufacturer’s recommendations. Efficiency of transfection was normalized by measuring β-Gal in cell lysates.
Western blot analysis
PGN preparations were boiled in Laemli SDS sample buffer and separated on 10% PAGE and transferred to nitrocellulose membrane. The rabbit antiserum #6344 raised against a peptide sequence common to several Staphylococcal and Streptococcal SAg [16
] was kindly donated by Dr. John Zabriskie, Rockefeller University, New York, NY, USA. It was used at 1:4000 dilution. The pooled human i.v. Ig (IVIG) was purchased from Blood Diagnostic (Irmo, SC, USA) and used at 1:500 dilution.
T cell proliferation
PBMC (6x105 cells/200 ml) were stimulated with PGN preparations (as seen and detailed in 
Fig. 3
legend) or the polyclonal mitogen PHA (1 µg/ml). After 72 h of culture, the cells were pulsed for the last 6 h with 1 µCi [3H] thymidine, harvested onto glass-fiber filters, and counted in a β-scintillation counter (Packard, Downers Grove, IL, USA). Each experiment was performed in quadruplicate and repeated at least three times.
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Figure 1. PGN but not TLR agonists induce IL-17. Human PBMC were stimulated for 20 h (A) or 6 h (B) with PG-Sa or the indicated agonists of TLR2 (Pam, FSL-1), TLR3 [poly(I:C)], TLR4 (LPS), and TLR7/TLR8 (R848). (A) IL-17 transcript and cytokine levels in culture supernatants were analyzed by RNase protection assay or ELISA. (B) Chemokine transcripts were analyzed by RNase protection assay. Agonists were used at the following concentration: LPS (10 ng/ml), PG-Sa (10 µg/ml), Pam3Cys (3 µg/ml), FSL-1 (100 ng/ml), poly(I:C) (30 µg/ml), R848 (1 µg/ml), PHA (1 µg/ml), PMA (20 ng/ml). One donor representative of several tested is shown. Ltn, Lymphotactin.
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Figure 2. Staphylococcal and Streptococcal PGN preparations induce IL-17 independently of TLR2-mediated signaling. (A) Human PBMC were stimulated for 6 h with PG-Sa (10 µg/ml), sPG-Ec (5 µg/ml), sPG-Sa (3 µg/ml), and PG-Sp (15 µg/ml). Cytokine and chemokine transcripts were analyzed by RNase protection assay. One experiment representative of three for each PGN preparation is shown. (B) The HeLa-TLR2 cell line was transiently transfected with an ELAM-luciferase reporter construct, CMV-CD14 and CMV-β-Gal (for normalization), and stimulated for 6 h with the PGN preparations (untreated or digested with PK). NF-B activation was measured by a luciferase assay. PG-Sa, 10 µg/ml; sPG-Sa, 10 µg/ml; PG-Sp, 40 µg/ml. One experiment representative of four is shown.
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Figure 3. IL-17 induction by PGN does not require inflammasome activation and is sensitive to PK digestion. (A) Human PBMC were stimulated for 6 h with PG-Sa (10 µg/ml), wild-type or listeriolysin mutant (LLO) Listeria monocytogenes (L.m.), MDP (30 µg/ml), and synthetic lipopeptide MALP2 (100 ng/ml). (B and C) PGN preparations were predigested with 300 ng PK for 2 h at 42°C before addition to PBMC cultures at the concentrations used in Figure 2A
. Cytokine transcripts were analyzed by RNase protection assay at 6 h, and IL-17 levels in culture supernatants at 20 h were measured by ELISA. PHA, 1 µg/ml. One experiment representative of two (A) or three (B) is shown. *, P < 0.05; **, P < 0.01.
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Statistical analysis
All data were expressed as mean ± SEM. Comparison of groups for statistical difference was done by using Student’s two-tailed t-test.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The discrepancy between the activity of PG-Sa and pure TLR2 agonists became evident when we analyzed the expression of IL-17, a cytokine produced by a subset of CD4 and CD8 T cells. As shown in Figure 1A , the mRNA of IL-17A and IL-17F was induced in human PBMC stimulated with PG-Sa but not with pure agonists of TLR2 (Pam3Cys, FSL-1), TLR3 [poly(I:C)], TLR4 (LPS), and TLR7/8 (R848). IL-17 transcripts were detected as early as 4 h poststimulation with PG-Sa and peaked at 20 h (not shown). IL-17 was readily detected in the conditioned supernatant of PBMC stimulated with PG-Sa but not other TLR agonists (Fig. 1A) . The inability of the other TLR agonists to induce IL-17 mRNA persisted, regardless of the dose used, and was not a result of insufficient cell stimulation, as the mRNA of several other cytokines (not shown) and chemokines (Fig. 1B) was robustly induced by all agonists. Thus, we concluded that IL-17 induction by PG-Sa does not correlate with TLR signaling.
Staphylococcal and Streptococcal PGN preparations induce IL-17 independently of TLR2-mediated signaling
Next, we extended this analysis to PGN preparations obtained from different vendors and extracted from different bacteria. We tested a commercial preparation of Streptococcus pyogenes PGN commonly used to elicit arthritis in animal models and preparations of soluble PGN obtained from E. coli or S. aureus. As shown in Figure 2A
, Streptococcal PGN (PG-Sp) or soluble Staphylococcal PGN (sPG-Sa) were potent inducers of IL-17 transcripts. In contrast, IL-17 message induction was not observed in cells stimulated with the soluble PGN from E. coli (sPG-Ec), which is a Nod1/Nod2 agonist. It should be noted that the insoluble PGN (PG-Sa) and soluble Staphylococcal PGN preparations are derived from different S. aureus strains and purchased from different vendors. The ability of PGN preparations to act as TLR2 agonists was tested using HeLa-TLR2, a cell line stably transfected with TLR2 [15
] (Fig. 2B)
. Robust, TLR2-dependent NF-B activation was detected only with PG-Sa. The Streptococcal PG-Sp induced weak NF-B activation only when used at high concentration (40 µg/ml), while sPG-Sa or sPG-Ec (not shown) was inactive, regardless of the dose used. As both PG-Sa preparations similarly induced IL-17, while only one activated cells through TLR2, we concluded that IL-17 induction does not require TLR2 signaling and that the component(s) of the PGN preparations that induced IL-17 is not a TLR2 agonist. Interestingly, the transcript of IL-23p19 was induced only by the PGN preparation that displayed potent TLR2 agonism (PG-Sa), in agreement with our previous study [15
]. On the other hand, the IP-10 transcript was induced by sPG-Sa but not PG-Sa (and only weakly by PG-Sp). This is surprising, as both Staphylococcal preparations induce IL-17 in a comparable way. We have previously shown that induction of IP-10 by different stimuli (TLR4/TLR3 agonists, IFN-β, IFN-) can be silenced by concomitant activation of TLR2 [18
]. Thus, it is possible that an unknown factor (see below) present in all PGN preparations has the potential to stimulate IP-10 expression, yet an IP-10 transcript can be detected only in cells stimulated with PGN preparations that are not contaminated with TLR2 agonists.
IL-17 induction by PGN preparations does not require inflammasome activation and is sensitive to PK digestion
Although not a powerful proinflammatory mediator, MDP, a subcomponent of PGN, can activate NF-B and the inflammasome (events that are mediated by Nod2 and Nalp3, respectively), leading to secretion of proinflammatory cytokines. Therefore, we tested whether induction of IL-17 by PGN preparations was due to MDP or required inflammasome activation. Figure 3A
shows that this is not the case. IL-17 was not induced by infection of cells with L. monocytogenes or treatment of cells with MDP (alone or in combination with TLR2 agonist MALP2), two treatments that lead to robust inflammasome activation [11
, 17
, 19
]. Moreover, induction of IL-17 by PG-Sa was not abolished by cotreatment with the caspase-1-specific inhibitor Z-YVAD. Thus, inflammasome activation or cytoplasmic PRR, such as Nod1/2 and Nalp3, are not involved in IL-17 induction by PGN.
Searching for clues about the nature of the IL-17-inducing factor, we found that while digestion with lysozyme did not affect PGN activity (not shown), digestion with PK destroyed the ability of PGN preparations to induce IL-17 (Fig. 3B) , indicating that the contaminant is a protein. Interestingly, PK treatment only marginally affected the ability of PG-Sa or PG-Sp to induce chemokines (Fig. 3B) or activate TLR2 signaling (Fig. 2B ; lipoprotein and the ability of LTA to activate TLR2 is not destroyed by proteases), yet it completely abolished the chemokine induction by sPG-Sa (which lacks TLR2 agonist activity). Thus, while IL-17 induction by all PGN preparations is mediated by a protein, chemokine induction occurs through different modalities among the different PGN preparations. Chemokine induction by PG-Sa and PG-Sp is mediated by factors resistant to protease treatment (TLR2 agonists), while induction by sPG-Sa is mediated by a protein and is independent of TLR2.
TCR signaling is required for IL-17 induction by PGN preparations
As IL-17 induction is known to require TCR engagement and as the IL-17-inducing factor contained in PGN is a protein, we hypothesized that SAg expressed by S. aureus or S. pyogenes may contaminate the commercial PGN preparations and be responsible for IL-17 induction. Several experimental data support this hypothesis. First, we noticed that although most chemokine and cytokine transcripts were induced at comparable levels among different PBMC donors, IL-17 induction varied greatly (not shown). This observation is consistent with our previous demonstration of a preferential presentation of SAg by certain HLA class II haplotypes [20
]. Second (Fig. 4
), we found that IL-17 induction (but not chemokine induction; not shown) by PGN was blocked by CsA, a drug that interferes specifically with TCR signaling and (not shown) by a MAPK p38 inhibitor or by bisindolylmaleimide, an inhibitor of atypical PKC such PKC-
, which is involved in TCR signaling. Thus, TCR signaling is required for IL-17 induction by PGN. Supporting the notion that PGN preparations may be able to trigger TCR signaling, T cell-specific cytokines IL-2, IL-4, IL-5, and IL-13 were induced by PGN or PHA, but not by TLR agonists.
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Figure 4. TCR signaling is required for IL-17 induction by PGN. Human PBMC were stimulated with PGN preparations in the presence or absence of cyclosporine (1 µg/ml) or TLR agonist, as detailed in the Figure 1
legend. Cytokine transcripts were analyzed by RNase protection assay. *, A nonspecific band derived from partial degradation of the IL-10 transcript. One experiment representative of three is shown.
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Figure 5. Staphylococcal and Streptococcal PGN preparations are contaminated by SAg. PGN preparations, untreated or digested with PK or lysozyme (Lys), were separated by PAGE, and the gel was stained with Coomassie blue dye (A) or transferred to nitrocellulose membranes and probed with rabbit anti-SAg serum #6344 (B) or pooled human IVIG (C). Purified rSpeC (500 ng, A; 50 ng, B) was loaded for size comparison. (B and C) PG-Sa (50 µg), 25 µg PG-Sp, or 20 µg sPG-Sa was loaded in each lane. MWM, Molecular weight markers; WB, Western blot; *, PK.
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Figure 6. Bacterial SAg induce IL-17. (A) Human PBMC were stimulated for 6 h with PG-Sa (10 µg/ml) or purified rSAg Staphylococcal TSST-1 (100 ng/ml), Streptococcal SpeC (250 ng/ml), and SmeZ (50 ng/ml). (B) Purified CD4+ T cells (7.8x106/ml) were stimulated for 6 h with TSST-1 (100 ng/ml) in the presence or absence of TLR2 agonist Pam (3 µg/ml) or APC (purified CD14+ monocytes, 7.8x105). Cytokine induction was analyzed by RNase protection assay or ELISA. One experiment representative of three is shown. *, P < 0.01.
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Figure 7. Staphylococcal and Streptococcal PGN preparations stimulate T cell proliferation. Human PBMC were stimulated with PGN preparations (at the concentration of Fig. 2A
), PHA (1.5 µg/ml), or a purified rSAg mixture. Tritiated thymidine incorporation was measured 72 h later. The experiment was repeated at least three times, and two representative responding donors are shown. *, P < 0.00001; **, P < 0.00005.
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Figure 8. Staphylococcal and Streptococcal PGN preparations stimulate expansion of T cells bearing specific Vβ elements. Human PBMC were stimulated with PGN preparations (undigested or PK-treated at the concentrations of Fig. 2A
) or PHA for 72 h. The percentage of CD3+ lymphocytes expressing selected Vβ elements was measured by FACS staining. Arrows indicate Vβ populations preferentially expanded by PGN treatment and the magnitude of the expansion (PGN:PHA ratio). One experiment representative of at least two responding donors is shown for each PGN preparation.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-17A and IL-17F are proinflammatory cytokines produced primarily by T cells (reviewed in ref. [27 ]). IL-17 plays an important role in host defense against some pathogens and is involved in several human autoimmune diseases. As IL-17 is produced by T cells and activates signaling events similar to those of other proinflammatory cytokines, it is considered as a molecule that links adaptive and innate immunity. The identification of the physiological stimuli that lead to IL-17 production by T cells is an area of intense research. It has been demonstrated that signaling through the TCR alone is sufficient to induce IL-17 expression, even in the absence of costimulation, IL-23, or APC [28 ], although these factors have been shown to contribute to IL-17 production [29 ]. Our results demonstrate that IL-17 induction by PGN preparations is mediated by the action of contaminating SAg rather than TLR or NLR agonists. The ability of SAg to potently induce IL-17, reported here for the first time, is in agreement with their ability to trigger TCR signaling in a considerable fraction of circulating T cells. To fulfill their stimulatory potential, SAg must simultaneously interact with the TCR and MHC II molecules on APC. Our finding that IL-17 induction by SAg occurs inefficiently in purified CD4 T cells, while it is increased by addition of APC is consistent with the accepted mechanism of action of SAg. Interestingly, in our experimental conditions, IL-23 does not appear to play a major role in IL-17 induction, as shown by the fact that sPG-Sa (Fig. 2) or purified SAg (Fig. 6) , which did not induce IL-23 expression, were still able to induce IL-17.
Several studies indicate that in humans and mice, IL-17 and the subset of Th cells (Th17) that expresses it play a role in allergic and autoimmune diseases such as airways hypersensitivity, psoriasis, inflammatory bowel disease, multiple sclerosis, and RA. The involvement of IL-17 in arthritis is particularly well documented (reviewed in ref. [30
]). By stimulating different cell types, IL-17 affects cartilage destruction and bone erosion directly through the induction of factors that regulate bone homeostasis (such as receptor activator of NF-B ligand and metalloproteases) and indirectly by potentiating the catabolic action of cytokines such as IL-1β and TNF-
. Elevated levels of IL-17 have been detected in the synovial fluid of patients with this disease [31
, 32
], and IL-17R-deficient mice are resistant to PGN-induced arthritis [33
, 34
], a widely used animal model of arthritis. In this experimental model [35
], injection of PGN into the joint of mice or rats triggers a form of polyarthritis with the hallmarks of the human disease, which includes infiltration of polymorphonuclear cells, CD4+ T cells, and macrophages, hyperplasia of the synovial lining layer, pannus formation, and moderate erosion of cartilage and bone. A large body of experimental evidence obtained using this and other animal models of arthritis points at bacterial products as possible etiologic factors of arthritis. Consistent with this notion, intestinally derived PGN can be detected in the synovial macrophages of RA patients [36
]. Interestingly, one of the PGN preparations routinely used to induce arthritis in this experimental model is the Streptococcal PGN preparation that we tested in the present study. This PGN preparation is also used to induce other types of experimental chronic inflammatory diseases, such as gastrointestinal inflammation, that resemble Crohn’s diseases. Based on our results, it is tempting to speculate that SAg that contaminate these PGN preparations may contribute, even substantially, to the arthritogenic activity of PGN. In support of the notion that SAg may play a role in the initiation or perpetuation of arthritis is the observation that SAg can reactivate PGN-induced arthritis [37
] and that arthritis can be experimentally induced by some mycoplasmas [38
, 39
], organisms that lack PGN but express SAg. It is also interesting to note that treatment of RA patients with a high dose of IVIG, which contains neutralizing anti-SAg antibodies, leads to improvement of the symptoms [40
]. On the other hand, it has been demonstrated that enzymatic digestion of PGN with the muralytic enzyme mutanolysin, which presumably should not affect possible contaminating SAg, abolishes induction of experimental arthritis, suggesting that the structure of PGN is also important [41
].
Although our results do not challenge the validity of the PGN-induced arthritis model or other experimental models where PGN triggers a specific pathology, they represent an important advancement for our understanding of the mechanistic aspects that are responsible for the ability of Gram-positive cell wall preparations to experimentally induce inflammatory diseases. Our results raise the possibility that cooperation and synergy among TLR agonists, NLR agonists, and SAg that are found in commercial PGN preparations may be the key to their potent inflammatory property. Future studies should determine the contribution of each of these components to the development of pathology.
Received August 31, 2007; revised October 9, 2007; accepted October 18, 2007.
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