Mycoplasma pneumoniae-Derived Lipopeptides Induce Acute ...

INFECTION AND IMMUNITY, Jan. 2008, p. 270–277 0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00955-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Mycoplasma pneumoniae-Derived Lipopeptides Induce Acute Inflammatory Responses in the Lungs of Mice䌤 Takashi Shimizu, Yutaka Kida, and Koichi Kuwano* The Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan Received 13 July 2007/Returned for modification 28 August 2007/Accepted 16 October 2007

The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributable to excessive immune responses. In this study, we investigated whether synthetic lipopeptides of subunit b of F0F1-type ATPase (F0F1-ATPase), NF-␬B-activating lipoprotein 1 (N-ALP1), and N-ALP2 (named FAM20, sN-ALP1, and sN-ALP2, respectively) derived from M. pneumoniae induce cytokine and chemokine production and leukocyte infiltration in vivo. Intranasal administration of FAM20 and sN-ALP2 induced infiltration of leukocyte cells and production of chemokines and cytokines in bronchoalveolar lavage fluid, but sN-ALP1 failed to do so. The activity of FAM20 was notably higher than that of sN-ALP2. FAM20 and sN-ALP2 induced tumor necrosis factor alpha (TNF-␣) through Toll-like receptor 2 in mouse peritoneal macrophages. Moreover, in the range of low concentrations of lipopeptides, FAM20 showed relatively high activity of inducing TNF-␣ in mouse peritoneal macrophages compared to synthetic lipopeptides such as MALP-2 and FSL-1, derived from Mycoplasma fermentans and Mycoplasma salivarium, respectively. These findings indicate that the F0F1-ATPase might be a key molecule in inducing cytokines and chemokines contributing to inflammatory responses during M. pneumoniae infection in vivo. tion, and we subsequently identified three lipoproteins responsible for NF-␬B activation. One was MPN602, known as subunit b of F0F1-type ATPase (F0F1-ATPase) (38), a diacylated lipoprotein. The activation of TLR signaling by F0F1ATPase was dependent on TLR1, TLR2, and TLR6. We identified two more lipoproteins, MPN611 and MPN162, and named them NF-␬B-activating lipoprotein 1 (N-ALP1) and N-ALP2, respectively. N-ALP1 and N-ALP2 activated TLR signaling through TLR1 and TLR2. Both N-ALP1 and NALP2 were assumed to be triacylated lipoproteins based on their activities (39). However, it has not been clear whether these lipoproteins induce inflammatory response in vivo or which lipoprotein is a key molecule for the inflammatory response. In this study, to examine the activity of synthetic lipopeptides derived from M. pneumoniae in an in vivo experimental system, lipopeptides were injected intranasally into mice. We found that partial synthetic lipopeptides derived from F0F1-ATPase, N-ALP1, and N-ALP2 (named FAM20, sN-ALP1, and sNALP2, respectively) induce chemokines and inflammatory cytokines followed by leukocyte infiltration in intranasal administration models in mice. The activity of FAM20 to induce the inflammatory response was approximately 100-fold higher than those of sN-ALP1 and sN-ALP2. The activity of FAM20 at low concentrations to induce inflammatory cytokines was higher than those of lipopeptides MALP-2 (31) and FSL-1 (37), derived from Mycoplasma fermentans and Mycoplasma salivarium, respectively.

Mycoplasmas are wall-less parasitic bacteria and the smallest organisms capable of self-replication (48). Mycoplasma pneumoniae causes primary atypical pneumonia, tracheobronchitis, pharyngitis, and asthma in humans (7, 19, 20). However, pathogenic agents such as endotoxin and exotoxin that cause such diseases have not been identified in M. pneumoniae. Adherence of invading mycoplasmas to the respiratory epithelium, localized host cell injury, and an overaggressive inappropriate immune response seem to contribute to the pathogenesis of M. pneumoniae infection (46). Recently, it has been reported that Toll-like receptors (TLRs) with the function of pattern recognition receptors play critical roles in early innate recognition and inflammatory responses of the host against invading microbes (1, 18). Among 10 TLR family members reported, TLR2, TLR4, TLR5, and TLR9 have been implicated in the recognition of different bacterial components. Peptidoglycan, lipoarabinomannan, zymosan, and lipoproteins from various microorganisms are recognized by TLR2 (2, 3, 24, 26, 43, 44, 47). On the other hand, lipopolysaccharide (LPS), bacterial flagellin, and bacterial DNA are recognized by TLR4, TLR5, and TLR9, respectively (9, 10, 13, 34). These TLR family members have been shown to activate nuclear factor ␬B (NF-␬B) via interleukin-1 (IL-1) receptor-associated signal molecules, including myeloid differentiation protein (MyD88), IL-1 receptor-activated kinase, tumor necrosis factor (TNF) receptor-associated factor 6, and NF-␬B-inducing kinase (27). We previously demonstrated that lipid-associated membrane proteins from M. pneumoniae can induce NF-␬B activa-

MATERIALS AND METHODS Synthesis of lipopeptides and LPS. A synthetic lipopeptide containing the N-terminal 20 amino acids of F0F1-ATPase derived from M. pneumoniae (FAM20) was synthesized by Bio Synthesis (Lewisville, TX). Lipopeptides sNALP1 and sN-ALP2 (Fig. 1) were synthesized as previously described (39). FSL-1 [S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF] (37) was purchased

* Corresponding author. Mailing address: 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Phone: 81-942-31-7548. Fax: 81-942-310343. E-mail: [email protected]. 䌤 Published ahead of print on 22 October 2007. 270

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above. Cytokine levels in the BALF were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, and BioSource, Camarillo, CA) according to the manufacturer’s instructions. The lower limits of detection for the ELISAs were as follows: 3.0 pg/ml for TNF-␣, 3.0 pg/ml for IL-6, 4.0 pg/ml for IL-10, 2.0 pg/ml for MCP-1, 1.5 pg/ml for MIP-2, and 3.0 pg/ml for MIP-3. Macrophage stimulation. One microgram of OK432 (Chugai Pharmaceutical, Tokyo, Japan) was injected into the peritoneal cavities of C57BL mice. Two days later, peritoneal exudate cells as macrophages were harvested and centrifuged. The cell pellets were suspended in serum-free medium optimized for macrophage culture (Gibco Invitrogen, Carlsbad, CA). Cells were allowed to adhere on 96-well culture plates for 2 h at 37°C with 5% CO2. Nonadherent cells were removed by washing with PBS, and the remaining adherent cells were stimulated with lipopeptides for 6 h. Statistical analysis. Results (expressed as means and standard deviations [SD]) were compared using one-way analysis of variance. The differences between groups were compared by multiple comparisons (Bonferroni t test) or Student’s t test. Differences were considered significant at a P value of ⬍ 0.05.

RESULTS

FIG. 1. Structure of synthetic lipopeptides. Di-O-palmitoyl S-(2,3dihydroxypropyl)-cysteinyl residues were N-terminally coupled to polypeptides. In the case of triacylated peptides, S-(2,3dihydroxypropyl)-cysteinyl residues were acylated with a third amidelinked palmitic acid.

from Invivogen (San Diego, CA). MALP-2 [S-(2,3-bispalmitoyloxypropyl)CGNNDESNISFKEK] was kindly provided by M. Matsumoto (Hokkaido University) (32, 36). LPS of Escherichia coli serotype O26:B6 was purchased from Sigma (St. Louis, MO). Mice. C57BL mice were purchased from Kyudo (Saga, Japan). TLR2 and TLR4 knockout (KO) mice, originally established by S. Akira (Osaka University) (1), were purchased from Oriental Bio Service (Kyoto, Japan). All experiments were conducted in compliance with institutional guidelines and have been approved by Kurume University. Lipopeptide administration in mice. C57BL mice were intranasally administered synthetic lipopeptides at 0.01 to 10 ␮g/mouse. At 6 to 72 h after administration, bronchoalveolar lavage fluid (BALF) was obtained by instilling 1 ml of phosphate-buffered saline (PBS) into the lungs and aspirating it from the tracheas of the mice using a tracheal cannula. The cells infiltrated in BALF were collected by centrifugation (3,000 rpm for 10 min), and the number of live cells in the infiltrates of BALF was determined by a trypan blue exclusion test. The supernatants were stored at ⫺80°C until determination of cytokine levels. Real-time PCR. C57BL mice were administered synthetic lipopeptides at 0.01 to 10 ␮g/mouse. At 6 to 72 h after administration, total RNA was obtained from whole lung tissue by using NucleoSpin (Clontech, Palo Alto, CA). Starting with 1 ␮g of total RNA, the synthesis of cDNA was performed by using an RNA PCR kit (Takara, Otsu, Japan). PCR was performed using iQ SYBR green supermix (Bio-Rad, Hercules, CA). The following primer sets were purchased from Takara: TNF-␣, 5-AAGCCTGTAGCCCACGTCGTA-3 (forward) and 5-GGC ACCACTAGTTGGTTGTCTTTG-3 (reverse); IL-6, 5-CCACTTCACAAGTC GGAGGCTTA-3 (forward) and 5-GCAAGTGCATCATCGTTGTTCATAC-3 (reverse); IL-10, 5-GACCAGCTGGACAACATACTGCTAA-3 (forward) and 5-GATAAGGCTTGGCAACCCAAGTAA-3 (reverse); monocyte chemoattractant protein 1 (MCP-1), 5-CCAACTCTCACTGAAGCCAGCTC-3 (forward) and 5-TTGGGATCATCTTGCTGGTGAA-3 (reverse); macrophage inflammatory peptide 2 (MIP-2), 5-GCGCTGTCAATGCCTGAAGA-3 (forward) and 5-TTTGACCGCCCTTGAGAGTG-3 (reverse); MIP-3, 5-ATGGGTACTGCT GGCTCACCTC-3 (forward) and 5-ACAAGCTTCATCGGCCATCTG-3 (reverse); and ␤-actin, 5-TGACAGGATGCAGAAGGAGA-3 (forward) and 5-G CTGGAAGGTGGACAGTGAG-3. All data were normalized to ␤-actin. Determination of cytokine levels in BALF. The levels of cytokines (TNF-␣, IL-6, and IL-10) and chemokines (MCP-1, MIP-2, and MIP-3) in BALF at 6 to 72 h after administration were determined. BALF was prepared as described

Leukocyte infiltration and dose-dependent release of cytokines and chemokines by FAM20. We previously demonstrated that a diacylated lipoprotein, MPN602, known as subunit b of F0F1-ATPase, induces NF-␬B through TLR1, TLR2, and TLR6. To test whether this lipoprotein induces inflammatory response in vivo, a synthetic lipopeptide (FAM20) containing the N-terminal 20 amino acids of F0F1-ATPase derived from M. pneumoniae was synthesized (Fig. 1). To elucidate whether this lipopeptide induces an inflammatory response in vivo, various concentrations of FAM20 were administered intranasally, and 24 h later, the number of infiltrated leukocytes in BALF was counted (Fig. 2A). Approximately 90% of infiltrated cells were found to be polymorphonuclear leukocytes (PMN) by hematoxylin staining (data not shown). The number of infiltrated PMN increased in a dose-dependent manner, and the dose of 1 ␮g of FAM20 showed maximum activity. To initially investigate whether FAM20 induces proinflammatory cytokines in vivo, RNA expression levels of TNF-␣ and IL-6 were measured by real-time PCR (Fig. 2B). The RNA expression levels of TNF-␣ and IL-6 were augmented dose dependently. As little as 0.1 ␮g of FAM20 was sufficient to induce RNA expression of proinflammatory cytokines. In contrast, the RNA expression level of anti-inflammatory cytokine IL-10 was low when 0.1 ␮g of FAM20 was administered. Ten micrograms of FAM20 was required to induce marked RNA expression of IL-10. To subsequently clarify whether cytokines such as TNF-␣, IL-6, and IL-10 are actually released, the concentrations of cytokines in BALF were measured by ELISA (Fig. 2C). The release of TNF-␣ and IL-6 increased dose dependently. One microgram of FAM20 showed maximum activity, and conversely, FAM20 at 10 ␮g decreased cytokine release. In contrast, the level of IL-10 release was not significant in the range of 0.1 to 10 ␮g of FAM20, although 10 ␮g of FAM20 induced marked RNA expression of IL-10 (Fig. 2B). Since FAM20 induced influx of PMN, the RNA expression levels of the mononuclear leukocyte-attracting CC chemokine MCP-1, MIP-3, and the neutrophil-attracting CXC chemokine MIP-2 were determined (Fig. 2D). Similar to the results for leukocyte infiltration (Fig. 2A), the RNA expression levels of MCP-1 and MIP-2 increased in a dose-dependent manner. The RNA expression of MCP-1 and MIP-2 at 1 ␮g of FAM20

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FIG. 2. Leukocyte infiltration and induction of cytokines and chemokines by FAM20. (A) C57BL mice were administered FAM20 at 0.01 to 10 ␮g/mouse. After 24 h, the number of cells infiltrated in BALF was determined by a trypan blue exclusion test. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 ␮g by Student’s t test. Two asterisks indicate that the P value is ⬍ 0.05 for a comparison with 0 ␮g by multiple comparison. (B and D) Total RNA was obtained from whole lung tissue administered FAM20. mRNAs were quantified by real-time PCR. Data are normalized to ␤-actin and expressed as a fold increase relative to the unstimulated control. All values represent the means and SD from a single experiment performed in triplicate, which was representative of three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 ␮g by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with 0 ␮g by multiple comparison. (C and E) Levels of cytokines and chemokines in BALF at 24 h after administration, determined using ELISA kits. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 ␮g by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with 0 ␮g by multiple comparison.

reached maximal levels, but an excess amount of FAM20 (10 ␮g) decreased the RNA expression levels. In contrast, the RNA expression levels of MIP-3 were almost constant in the range of 0.1 to 10 ␮g of FAM20. To investigate actual chemokine release, the concentrations of chemokines in BALF were measured by ELISA (Fig. 2E). Correlating with the result of RNA expression (Fig. 2D), the release of MCP-1, MIP-2, and MIP-3 was maximal when 1 ␮g of FAM20 was administered. Time kinetics of leukocyte infiltration and release of cytokines and chemokines by FAM20. To investigate the time kinetics of the inflammatory response induced by FAM20, 1 ␮g of FAM20 was administered intranasally. After 6, 12, 24, 48, and 72 h, the number of infiltrated leukocytes in BALF was counted. As shown in Fig. 3A, the number of the infiltrated PMN reached a maximum at 24 h. To examine the time kinetics of the induction of cytokine production by FAM20, the RNA expression levels of TNF-␣,

IL-6, and IL-10 were determined by real-time PCR (Fig. 3B). The expression levels of proinflammatory cytokines TNF-␣ and IL-6 were maximal at 48 h and 12 h, respectively. The maximal expression level of anti-inflammatory cytokine IL-10 was observed at 48 h. Thus, there were no apparent differences in the time kinetics between pro- and anti-inflammatory cytokines. To subsequently evaluate the release of such cytokines as TNF-␣, IL-6, and IL-10, the concentrations of cytokines in BALF were measured by ELISA (Fig. 3C). The release of TNF-␣ was maximal at 48 h after administration, consistent with the result for the RNA levels of TNF-␣ (Fig. 3B). The release of IL-6 was detected at as early as 6 h and reached a maximum at 48 h. Unexpectedly, the release of IL-10 was below the level of detection at all times, although the RNA expression of IL-10 was observed after 6 h (Fig. 3B). To further test the time kinetics of chemokine induction, the RNA expression levels of chemokines MCP-1, MIP-2, and

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FIG. 3. Time kinetics of cytokine and chemokine release by FAM20. (A) C57BL mice were administered FAM20 at 1.0 ␮g/mouse. At 6 to 72 h after administration, the cells infiltrated in BALF were determined by a trypan blue exclusion test. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 h by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with 0 h by multiple comparison. (B and D) Total RNA was obtained from whole lung tissue administered FAM20. mRNAs were quantified by real-time PCR. Data are normalized to ␤-actin and expressed as a fold increase relative to the unstimulated control. All values represent the means and SD from a single experiment in triplicate, which was representative of three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 h by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with 0 h by multiple comparison. (C and E) Levels of cytokines and chemokines in BALF at the indicated hours after administration were determined using ELISA kits. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with 0 h by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with 0 h by multiple comparison.

MIP-3 were measured (Fig. 3D). The RNA expression levels of the three chemokines showed similar kinetics with a maximum at 12 h. To investigate the actual release of chemokines, the concentrations of chemokines in BALF were measured by ELISA (Fig. 3E). The release of MCP-1 and MIP-3 was maximal at 48 h and 24 h, respectively, after administration. Interestingly, MIP-2 was detected at as early as 6 h with a maximum level, followed by decline. Leukocyte infiltration and cytokine and chemokine induction by sN-ALP1 and sN-ALP2. We previously reported that triacylated lipoproteins MPN611 and MPN162 induce NF-␬B activation, and we named these lipoproteins N-ALP1 and NALP2, respectively. We also synthesized lipopeptides of NALP1 and N-ALP2 (sN-ALP1 and sN-ALP2, respectively) (Fig. 1). To test whether N-ALP1 and N-ALP2 induce an inflammatory response in vivo, sN-ALP1 and sN-ALP2 were administered intranasally to mice, and after 24 h, the number of infiltrated leukocytes was counted. No infiltrated cells were

observed when 1 ␮g each of sN-ALP1 and sN-ALP2 was administered (data not shown). When 10 ␮g of sN-ALP2 was administered, the number of infiltrated cells was slightly increased (significant by Student’s t test but not significant by multiple comparison), although the level of the infiltration was lower than that with FAM20 at 10 ␮g (Fig. 4A). However, no infiltrated cells were observed when 10 ␮g of sN-ALP1 was administered. Thus, the activity of both lipopeptides to induce influx of PMN was apparently lower than that of FAM20. To investigate the induction of proinflammatory cytokines by sN-ALP1 and sN-ALP2, the RNA expression levels of TNF-␣ and IL-6 were determined by real-time PCR (Fig. 4B). sN-ALP2 remarkably augmented the expression levels of both cytokines. In contrast, the expression levels induced by sNALP1 were lower than those induced by sN-ALP2. Interestingly, the RNA expression levels of anti-inflammatory cytokine IL-10 were significantly increased by both sN-ALP1 and sNALP2. To subsequently determine the actual release of cytokines,

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FIG. 4. Leukocyte infiltration and induction of cytokines and chemokines by sN-ALP1 and sN-ALP2. (A) C57BL mice were administered each lipopeptide at 10 ␮g/mouse. At 24 h after administration, the number of cells infiltrated in BALF was determined by a trypan blue exclusion test. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with PBS by Student’s t test. Two asterisks indicate that the P value is ⬍ 0.05 for a comparison with PBS by multiple comparison. (B and D) Total RNA was obtained from whole lung tissue administered each lipopeptide. mRNAs were quantified by real-time PCR. Data are normalized to ␤-actin and expressed as a fold increase relative to the unstimulated control. All values represent the means and SD from a single experiment in triplicate, which was representative of three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with PBS by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with PBS by multiple comparison. (C and E) Levels of cytokines and chemokines in BALF at 24 h after administration were determined using ELISA kits. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). An asterisk indicates that the P value is ⬍0.05 for a comparison with PBS by Student’s t test. Two asterisks indicate that the P value is ⬍0.05 for a comparison with PBS by multiple comparison.

the concentrations of cytokines in BALF were measured (Fig. 4C). When sN-ALP2 was administered, low levels of TNF-␣ and IL-6 were detected in BALF (significant by Student’s t test but not significant by multiple comparison). In contrast, the administration of sN-ALP1 failed to induce cytokine release. FAM20 as a positive control markedly induced the release of both cytokines. Interestingly, the release of IL-10 in BALF was not detected despite the expression of IL-10 mRNA induced by FAM20, sN-ALP1, and sN-ALP2 (Fig. 4B). To further examine chemokine induction by sN-ALP1 and sN-ALP2, the RNA expression levels of MCP-1, MIP-2, and MIP-3 were determined by real-time PCR (Fig. 4D). Elevated RNA expression of three chemokines by sN-ALP2 was observed, but the RNA expression levels of the three chemokines were lower than those of FAM20. Nevertheless, the administration of sN-ALP1 failed to induce RNA expression of these chemokines. To determine the actual release of chemokines, the levels of chemokines in BALF were measured (Fig. 4E). When sN-

ALP2 was administered, low levels of MIP-3 were detected (significant by Student’s t test but not significant by multiple comparison), but the release levels of MCP-1 and MIP-2 were not significant. FAM20 as a positive control strikingly induced the release of these chemokines. In contrast, the administration of sN-ALP1 failed to induce the release. TLR2-dependent cytokine and chemokine induction. Lipoproteins derived from various microorganisms are reported to be recognized by TLR2 (2, 3, 24, 26, 43–45, 47). Thus, the TLR dependency of the inflammatory responses induced by FAM20, sN-ALP1, and sN-ALP2 was examined. Peritoneal macrophages from TLR2 and TLR4 KO mice were stimulated with lipopeptides, followed by measurement of TNF-␣ levels (Fig. 5). When peritoneal macrophages from wild-type (WT) mice were stimulated with lipopeptides and LPS, TNF-␣ was released from the macrophages stimulated with FAM20, sNALP1, sN-ALP2, and LPS, although the levels of TNF-␣ induced by sN-ALP1 were lower than those induced by other stimulants. When macrophages from TLR4 KO mice were


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FIG. 5. TLR2-dependent cytokine and chemokine induction. A total of 3 ⫻ 105 peritoneal macrophages/well obtained from TLR KO mice were cultured in a 96-well plate and stimulated with lipopeptides or LPS for 6 h. Levels of TNF-␣ in the culture supernatants were measured using ELISA kits. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). Two asterisks indicate that the P value is ⬍ 0.05 for a comparison with the WT by multiple comparison.

stimulated with lipopeptides, TNF-␣ release from the macrophages stimulated with FAM20 and sN-ALP2 partially decreased compared to that from WT macrophages. TNF-␣ release by LPS was strikingly suppressed in TLR4 KO macrophages. In contrast, TNF-␣ release induced by FAM20, sN-ALP1, and sN-ALP2 was considerably impaired in macrophages from TLR2 KO mice. Comparison of activity levels of lipopeptides derived from various mycoplasmas. It has been reported that synthetic lipopeptides MALP-2 and FSL-1, derived from M. fermentans and M. salivarium, respectively, stimulate the host immune response (31, 37). To compare the activities of these lipopeptides to induce an inflammatory response, peritoneal macrophages from WT mice were stimulated with various concentrations of synthetic lipopeptides, including MALP-2, FSL-1, FAM20, sN-ALP1, and sN-ALP2. All of the lipopeptides induced TNF-␣ release dose dependently, but the activities of sN-ALP1 and sN-ALP2 were significantly lower than those of other lipopeptides (Fig. 6). At high concentrations (more than 1 nM), MALP-2, FSL-1, and FAM20 showed similar activity. Neither MALP-2 nor FSL-1 at low concentrations of 0.01 and 0.1 nM induced TNF-␣ induction, but FAM20 at a comparable dose still had activity to induce TNF-␣ (P ⬍ 0.05). DISCUSSION M. pneumoniae causes primary atypical pneumonia, asthma, and other respiratory diseases in humans (7, 19, 20). Adherence of invading mycoplasmas to the respiratory epithelium and localized host cell injury are thought to be responsible for the pathogenesis of M. pneumoniae infections. Although several toxins, such as hydrogen peroxide, hemolysin, and community-acquired respiratory distress syndrome toxin, have been reported (6, 15, 41), excessive immune responses are thought to participate in the pathogenesis (35, 46). However, such factors associated with the induction of the immune response have not been identified in M. pneumoniae. We previously demonstrated that three lipoproteins, F0F1ATPase, N-ALP1, and N-ALP2, derived from M. pneumoniae induce NF-␬B activation through TLR2 (38, 39). However, it

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FIG. 6. Comparison of lipopeptides derived from various mycoplasmas. A total of 3 ⫻ 105 peritoneal macrophages/well obtained from WT mice were cultured in a 96-well plate and stimulated with the indicated concentrations of lipopeptides for 6 h. Levels of TNF-␣ in the supernatants were measured using ELISA kits. Representative data from three separate experiments are shown. All values represent the means and SD from three independent experiments (n ⫽ 3 in each group). Two asterisks indicate that the P value is ⬍0.05 for a comparison with MALP-2 and FSL-1 by multiple comparison.

has not been clear whether these lipoproteins induce inflammatory response in vivo or which lipoprotein is a key molecule for the inflammatory response. In this study, we demonstrated that the synthetic lipopeptides FAM20 and sN-ALP2 (derived from F0F1-ATPase and N-ALP2, respectively) induce chemokines and inflammatory cytokines followed by PMN infiltration in intranasal administration models in mice. Our data are consistent with earlier reports that a high percentage of neutrophils are present in BALF of patients with M. pneumoniae and that the levels of various cytokines are elevated in BALF (17, 23, 33, 50). These findings suggest that lipoproteins of M. pneumoniae contribute to the pathogenesis of M. pneumoniae infection. Although 46 putative lipoproteins were found from the genome sequence of M. pneumoniae (11), we have so far purified three lipoproteins, F0F1-ATPase, N-ALP1, and N-ALP2, that induce inflammatory responses (38, 39). Interestingly, the activity of F0F1ATPase to induce the inflammatory response seemed to be much higher than those of N-ALP1 and N-ALP2. We cannot rule out the possibility that the remaining 43 putative lipoproteins have NF-␬B inducing activity in vivo, but recently, diacylated lipopeptides synthesized from the consensus sequence of a putative paralogous lipoprotein group characteristic of M. pneumoniae (designated M. pneumoniae paralogous lipoprotein 1 [MPPL-1]) were reported to activate NF-␬B-dependent gene transcription (14). MPPL-1 showed much lower induction of proinflammatory cytokines than FSL-1 and MALP-2, derived from M. salivarium and M. fermentans, respectively. However, F0F1-ATPase does not contain the consensus sequence of a putative paralogous lipoprotein group such as MPPL-1. In fact, FAM20 showed the remarkable activity of inducing TNF-␣ production even at low concentrations compared to FSL-1 and MALP-2. The precise reasons for the relatively higher activity of FAM20 are unclear; however, the amino acid sequences of lipoproteins would play essential roles in the expression of the activity (5). These findings strongly suggest that F0F1-ATPase is a key molecule responsible for the induction of the inflammatory response in vivo. These findings may

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also explain the mechanism by which M. pneumoniae infection causes severe pathogenesis compared to M. fermentans and M. salivarium. Unexpectedly, the activity of triacylated lipopeptides sNALP1 and sN-ALP2 to induce the inflammatory response in mice model was much lower than that of diacylated lipopeptide FAM20, although sN-ALP1 and sN-ALP2 showed significant NF-␬B induction activity in a human kidney cell 293T cell model (39). This finding is in line with the report that the activity of diacylated MALP-2 to induce nitrogen oxide in mice is approximately 100-fold higher than that of triacylated MALP-2 (30). It is widely known that triacylated lipoproteins are recognized by TLR1 and TLR2, whereas diacylated lipoproteins are recognized by TLR2 and TLR6. Both TLR1 and TLR6 were reported to be expressed at almost comparable levels in cells, including macrophages, T cells, NK cells, B cells, and dendritic cells (DC) (12). Considering these findings, diacylated lipoproteins might bind more tightly to TLR6 than triacylated lipoproteins bind to TLR1. Indeed, the diacylated lipoprotein F0F1-ATPase induced the prominent activation of NF-␬B in human 293T cells through TLR1 as well as TLR6 (38). This evidence indicates that the activity of the triacylated lipopeptides to induce cytokine is somewhat lower than that of the diacylated lipopeptides in mouse models. Our data also provide an explanation for the leukocyte infiltration and inflammatory response in M. pneumoniae infection. In our experimental system, early induction (within 6 h after administration) of the neutrophil-attracting CXC chemokine MIP-2 was observed, leading to the influx of leukocytes and the induction of proinflammatory cytokines and mononuclear leukocyte-attracting CC chemokines (24 to 48 h). MIP-2 is reported to be a key chemokine regulating the infiltration of PMN in other bacterial infections, such as infections with Klebsiella pneumoniae, Pseudomonas aeruginosa, and Shigella flexneri (4, 16, 40). These findings may suggest that that F0F1ATPase induces certain factors, including MIP-2, that are responsible for the influx of neutrophils and that the concomitant induction of cytokines results in the inflammatory responses in lungs infected with M. pneumoniae. Curiously, FAM20, sN-ALP1, and sN-ALP2 at 10 ␮g each induced RNA expression of IL-10 but were insufficient for IL-10 release, suggesting that another signal may be required for sufficient IL-10 production. It is noteworthy that the C-type lectin DC-SIGN modulates TLR signaling via Raf-1 kinasedependent acetylation of NF-␬B and enhances the production of IL-10 by DC (8). IL-10 inhibits multiple macrophage and DC effector functions and plays a critical role in limiting tissue injury during infections and in preventing autoimmunity by limiting the duration and intensity of immune and inflammatory reactions (28). Therefore, insufficient IL-10 induction may be responsible for the excessive immune response in M. pneumoniae infection. However, further study is required to explain the mechanism of the immune response. Our findings also suggest that F0F1-ATPase might be a potential therapeutic target. For examples, F0F1-ATPase and its derivatives might be suitable candidates as antigens for vaccination, causing the induction of specific antibodies capable of inhibiting TLR signaling. Moreover, F0F1-ATPase with modified acyl chains would be a potential antagonist for TLR signaling, because unsaturated fatty acids have been reported to

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inhibit TLR2 signaling induced by lipopeptides (21, 22). Recently, increasing numbers of macrolide-resistant M. pneumoniae strains have been reported (25, 29, 42, 49), indicating that chemotherapy may be difficult in the future. Hence, lipoproteins such as F0F1-ATPase might be potential molecules in the development of alternative weapons for the prevention and treatment of M. pneumoniae infection. ACKNOWLEDGMENTS We thank M. Matsumoto for providing MALP-2 and T. Kakuma (Department of Bio-statistics, Kurume University School of Medicine) for assistance with the statistical analysis. This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES 1. Akira, S., and K. Takeda. 2004. Toll-like receptor signaling. Nat. Rev. Immunol. 4:499–511. 2. Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, and A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285:736–739. 3. Brightbill, H. D., D. H. Libraty, S. R. Krutzik, R. B. Yang, J. T. Belisle, J. R. Bleharski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, P. J. Brennan, B. R. Bloom, P. J. Godowski, and R. L. Modlin. 1999. Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Science 285:732–736. 4. Broug-Holub, E., G. B. Toews, J. F. van Iwaarden, R. M. Strieter, S. L. Kunkel, R. Paine III, and T. J. Standiford. 1997. Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival. Infect. Immun. 65:1139–1146. 5. Buwitt-Beckmann, U., H. Heine, K. H. Wiesmuller, G. Jung, R. Brock, S. Akira, and A. J. Ulmer. 2005. Toll-like receptor 6-independent signaling by diacylated lipopeptides. Eur. J. Immunol. 35:282–289. 6. Cohen, G., and N. L. Somerson. 1967. Mycoplasma pneumoniae: hydrogen peroxide secretion and its possible role in virulence. Ann. N. Y. Acad. Sci. 143:85–87. 7. Gil, J. C., R. L. Cedillo, B. G. Mayagoitia, and M. D. Paz. 1993. Isolation of Mycoplasma pneumoniae from asthmatic patients. Ann. Allergy 70:23–25. 8. Gringhuis, S. I., J. den Dunnen, M. Litjens, B. van Het Hof, Y. van Kooyk, and T. B. Geijtenbeek. 2007. C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB. Immunity 26:605–616. 9. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099–1103. 10. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740–745. 11. Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B. C. Li, and R. Herrmann. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24:4420–4449. 12. Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann. 2002. Quantitative expression of Toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531–4537. 13. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, and S. Akira. 1999. Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749–3752. 14. Into, T., J. Dohkan, M. Inomata, M. Nakashima, K. Shibata, and K. Matsushita. 2007. Synthesis and characterization of a dipalmitoylated lipopeptide derived from paralogous lipoproteins of Mycoplasma pneumoniae. Infect. Immun. 75:2253–2259. 15. Kannan, T. R., and J. B. Baseman. 2006. ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens. Proc. Natl. Acad. Sci. USA 103:6724–6729. 16. Kernacki, K. A., R. P. Barrett, J. A. Hobden, and L. D. Hazlett. 2000. Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection. J. Immunol. 164:1037–1045. 17. Koh, Y. Y., Y. Park, H. J. Lee, and C. K. Kim. 2001. Levels of interleukin-2, interferon-gamma, and interleukin-4 in bronchoalveolar lavage fluid from patients with Mycoplasma pneumonia: implication of tendency toward increased immunoglobulin E production. Pediatrics 107:E39.

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