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Myeloid differentiation factor 2 (MD-2) is a secreted glycoprotein that assembles with Toll-like receptor 4 (TLR4) to form a functional signaling receptor for bacterial lipopolysaccharide (LPS). In this study we have identified a novel alternatively spliced isoform of human MD-2, termed MD-2 short (MD-2s), which lacks the region encoded by exon 2 of the MD-2 gene. Similar to MD-2, MD-2s is glycosylated and secreted. MD-2s also interacted with LPS and TLR4, but failed to mediate LPS-induced NF-κB activation and interleukin-8 production. We show that MD-2s is upregulated upon IFN-γ, IL-6 and TLR stimulation and negatively regulates LPS-mediated TLR4 signaling. Furthermore, MD-2s competitively inhibited binding of MD-2 to TLR4. Our study therefore pinpoints a mechanism that may be employed to regulate TLR4 activation at the onset of signaling and identifies MD-2s as a potential therapeutic candidate to treat human diseases characterized by an overly exuberant or chronic immune response to LPS.
Detection of microbial pathogens and instigation of an appropriate innate and subsequent adaptive immune response is highly reliant on Toll-like receptors (TLRs) (1). TLR4 is one of the most widely studied of the family and recognizes a varied repertoire of ligands, such as heat shock protein 60 (2), respiratory syncytial virus fusion protein (3) and lipopolysaccaride (LPS) (4–8) a major component of the outer membrane of Gram-negative bacteria. Host sensitivity to LPS is enhanced by the accessory proteins, LPS-binding protein (9) and CD14 (10), but for LPS recognition to occur, TLR4 requires the co-receptor myeloid differentiation factor (MD)-2 (11–13). Upon LPS binding, a receptor multimer composed of two copies of the TLR4-MD-2-LPS complex is formed (14), which triggers a downstream signaling cascade, culminating in the activation of transcription factors such as nuclear factor (NF)-κB and the interferon regulatory factors (IRFs), which in turn induce various immune and inflammatory genes.
Tight regulation of TLR4 signaling is imperative in order to prevent an overactivated immune response that could contribute to the pathogenesis of autoimmune, chronic inflammatory and infectious diseases, such as diabetes (15), asthma (16) and sepsis (4). One method of downregulating TLR4 signaling involves the production of inhibitory isoforms, by alternatively splicing specific genes encoding essential signaling components. To date, several such splice variants have been identified, examples include smTLR4 (17, 18), myeloid differentiation factor 88S (MyD88S) (19), TRAM adaptor with GOLD domain (TAG) (20) and murine MD-2B (21).
Here we report the identification and characterization of a novel alternatively spliced isoform of human MD-2, that we have called MD-2 short (MD-2s). Similar to full-length MD-2, this protein was glycosylated and secreted. However, despite its ability to interact with TLR4 and LPS, MD-2s failed to mediate NF-κB activation and interleukin 8 (IL-8) production following LPS exposure. We also determined that MD-2s competitively inhibited binding of full length MD-2 to TLR4 and identified this short isoform as a negative regulator of LPS-mediated TLR4 activation. We show that MD-2s is an inducible protein that may function as a negative feedback inhibitor. Our results therefore define a novel mechanism used by human isoform of MD-2 that may curtail excessive activation of the innate immune response at the initiation of the TLR4 signal transduction pathway.
Immortalized HMECs were cultured in MCDB-131 medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 100 μg/ml penicillin and streptomycin. The HEK293 cell line, mouse RAW 264.7 macrophage cell line, mouse aortic endothelial cells (MAEC) were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM glutamine. LPS (TLRGrade; Alexis). Biotin-LPS (Ultrapure, Invivogen).
Total cellular RNA was isolated from cells using the RNeasy mini kit (Qiagen) and treated with DNase. RNA from human lung, pancreas, thymus, kidney, spleen, liver, heart, and placenta was purchased from Ambion. Following reverse transcription with Omniscript cDNA synthesis kit (Qiagen), PCR analysis was performed using primers specific for human MD-2 (5′-ATGTTACCATTTCTGTTT-3′, 5′-CTAATTTGAATTAGGTTG-3′) or mouse MD-2 (5′-TCTGCAACTCCTCCGATG-3′, 5′-GGCGGTGAATGATGGTGA-3′). The PCR was performed using Taq DNA polymerase (Invitrogen). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. For quantitative RT-PCR the following primer and probe set was used to detect MD-2s: 5′-ATT GGG TCT GCA ACT CAT CC-3′, 5′-TTC TTT GCG CTT TGG AAG AT-3′ and 5′-CAC CTA CTG TGG GAG AGA TTT AAA GCA-3′. The comparative cycling threshold method (ΔΔCT) was used for relative quantification compared to untreated samples after normalization with GAPDH expression.
HEK293 cells were seeded into 100 mm dishes (1.5×106) 24 h prior to transfection. Transfections were performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. For co-immunoprecipitations, 4 μg of each construct was transfected. For competition experiments where the effect of increasing MD-2s expression on complex formation between MD-2 and TLR4 was examined, 3 μg of each signaling molecule expression plasmid was transfected in the presence of increasing amounts (10 μg or 15μg) of the MD-2s expression plasmid. The total amount of DNA in each sample was kept constant by using empty vector cDNA. Cells were harvested 24 h following transfection in 600 μl of lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 containing protease inhibitor cocktail, and 1 mM sodium orthovanadate). For immunoprecipitations, the indicated antibodies were incubated with the cell lysates for 2 h or overnight at 4°C. Subsequently, Trueblot™ IgG beads were added and the samples were incubated at 4°C for 1 h. The immune complexes were then washed and the associated proteins were eluted from the beads by boiling in 35 μl of sample buffer, and then fractionated by SDS-PAGE. For immunoblotting, primary antibodies were detected using horseradish peroxidase–conjugated secondary antibodies, followed by enhanced chemiluminescence (Amersham Biosciences).
HEK293 cells were transiently transfected with the expression vectors noted in combination with constructs encoding the NF-κB-luciferase reporter gene, and the phRL-TK reporter gene to normalize for transfection efficiency. In all cases, total DNA concentration was kept constant by supplementation with empty vector control. Following overnight incubation, cells were stimulated for 6 h with 50 ng/ml LPS, lysed and then luciferase activity was measured. For the inhibition studies, HEK293 cells were transfected with a constant amount (3 μg) of MD-2 expressing plasmid alone or in combination with increasing concentrations of a plasmid expressing MD-2s (5 μg, 10 μg, 20 μg). In all cases, total DNA concentration was kept constant by supplementation with empty vector control. Supernatants derived from these cells (5 ml final volume) were then transferred (100 μl per well) to HEK293 cells stably transfected with TLR4 and a NF-κB reporter gene. These supernatant-treated cells were then stimulated with LPS (5 ng/ml) for 6 h, lysed and then luciferase activity was measured. Data are shown as mean ± S.D. of three or more independent experiments and are reported as a percentage of LPS-stimulated NF-κB promoter activity.
After LPS stimulation, supernatants were collected and the production of human IL-8 was measured with an ELISA kit following the manufacturers directions (R&D Systems).
HEK293T cells were retrovirally transduced to generate a cell line stably expressing TLR4-mCitrine. TLR4-mCitrine cells were seeded into 12-well dishes and transfected with 300 ng of plasmid encoding Myc-tagged MD-2 or MD-2s using GeneJuice lipofection reagent (Novagen) and cultured overnight. Cells were dislodged by scraping, washed once with cold PBS and incubated with a 1:20 dilution of anti-Myc Alexa 647-conjugated antibody (AbD Serotec) at 4 °C for 20 min. Cells were washed four times with PBS and fluorescence was assessed with an LSR II flow cytometer (BD Biosciences) running FACS Diva software (BD Biosciences). Data was processed using FlowJo software v8.6.3 (Tree Star).
TLR4-mCitrine cells were cultured on glass-bottom confocal dishes (MatTek) coated with collagen. After 24 h of culture, cells were transfected with up to 300 ng of plasmid encoding myc-tagged MD-2 or MD-2s using GeneJuice lipofection reagent (Novagen) and cultured for approximately 24 h. Cells were stained with anti-myc Alexa-647 antibody (AbD Serotec) in culture medium at 4 °C for 20 min. Cells were gently washed three times with PBS and culture medium was replaced. Cells were subsequently imaged at room temperature using an SP2 AOBS confocal laser scanning microscope (Leica Microsystems) running LCS software (Leica Microsystems).
We examined the expression profile of MD-2 in human monocytic THP-1 cells by RT-PCR analysis and detected two cDNA products, suggesting that the human MD-2 gene is alternatively spliced (Fig. 1a). RT-PCR amplification using cDNA derived from human dermal microvascular endothelial cells (HMECs) also yielded two fragments. Sequencing of the larger cDNA fragment determined that it corresponded to the published sequence of full-length human MD-2. Sequence analysis of the smaller cDNA fragment, subsequently referred to as MD-2s, revealed that this was a novel splice variant of human MD-2, lacking the region encoded by exon 2 (Fig. 1b and 1c). The open reading frame of MD-2s consists of 390 bp, which translates into a predicted protein of 130 amino acids. Excision of exon 2 resulted in an in-frame deletion of 90 bp and an amino acid substitution (D38G) at the junction between exons 2 and 3. The existence of MD-2s was further confirmed given that a full-length mRNA sequence, deposited in the NCBI database, corresponded to MD-2s (Accession code: BM918324). Table I depicts the 3′ and 5′ splice-site sequences at the intron-exon boundaries for MD-2 and MD-2s.
We also examined the expression profile of murine MD-2 by performing RT-PCR on cDNA derived from the murine monocytic cell line, RAW246.7, and mouse aortic endothelial cells (MAECs). Using primers specific for murine MD-2, only the larger RT-PCR product was detected (Fig. 1d, lanes 1 and 2 respectively). To confirm that the absence of the smaller fragment was not specific to the murine cell lines selected, we also amplified cDNA from murine bone marrow derived dendritic cells and liver tissues obtained from C57BL/6 mice. Again only the larger cDNA fragment was observed, suggesting that MD-2s is not expressed in mice (Fig. 1d, lanes 3 and 4). Furthermore, of the four murine MD-2 splice variants that have been deposited in the NCBI database, exon 2 is transcribed in all isoforms identified.
To determine why the human but not the mouse MD-2 gene alternatively skips exon 2, we compared the gene structures and sequences of the two species. Both the human and murine MD-2 genes are organized into five exons and four introns, and each species encodes a predicted full-length MD-2 protein of 160 amino acids. Alignment of the human and mouse genomic regions revealed that the exons and coding sequences were well conserved. However, analysis of the non-coding regions revealed a number of differences. In particular, it was noted that intron 1 of human MD-2 is composed of 13241 base pairs, while the mouse has 3064. The longer intron 1 may increase the probability for alternative lariat formation during the splicing process of human MD-2. In addition, after comparing the sequences at the 3′ end of intron 1, we found that murine MD-2 has more pyrimidines than its human ortholog, which may lead to a more stable lariat formation in murine MD-2, thereby preventing alternative splicing of exon 2 in this species.
To further characterize the expression profile of human MD-2s, we performed RT-PCR analysis on a variety of human tissues. As shown in Fig. 2a, MD-2s mRNA was expressed in all tissues examined. We also observed that although the ratio between MD-2 and MD-2s varies in different tissues, full-length MD-2 is the predominant form detected. Previous studies have indicated that MD-2 mRNA is upregulated following exposure to interferon-gamma (IFN-γ) (22, 23) or IL-6 (13). We therefore investigated what effect these stimuli would have on MD-2s mRNA levels. RNA was extracted from THP-1 cells stimulated with IFN-γ- or IL-6, and quantitative RT-PCR was performed with primers specific for MD-2s and the reference gene GAPDH. Notably, we observed induction of MD-2s mRNA following treatment with both IL-6 and IFN-γ (Fig. 2b). We next examined what effect LPS stimulation would have on MD-2s expression. Interestingly, MD-2s expression was also upregulated in response to LPS (Fig. 2c). This suggests that MD-2s may play an important regulatory role during immune responses and TLR signaling.
In order to further characterize MD-2s, we amplified the smaller RT-PCR fragment and cloned it into an expression vector encoding for the Myc tag. When this plasmid was transfected into human embryonic kidney (HEK293) cells, overexpressed MD-2s migrated with different electrophorectic mobilities following SDS-PAGE analysis (Fig. 2d, lane 1). MD-2 displays a similar expression profile due to N-linked glycosylations at positions Asn26 and Asn114 (24). These residues are still present in MD-2s, but given that it lacks 30 amino acids, its tertiary structure was likely to be different than that of MD-2, which could result in occlusion of these known glycosylation sites. Nevertheless, we found that following treatment with the Peptide-N-glycosidase F (PNGase F), the slowest migrating forms of MD-2s were no longer evident in the PNGase F-treated sample (Fig. 2d, lane 2). Thus, MD-2s is also a glycoprotein.
MD-2 belongs to the MD-2-related lipid recognition (ML) family, the signature sequence of which is a secretion signal (25). This signal is located at the N-terminus of MD-2, and studies have shown that MD-2 is secreted (26, 27). MD-2s also contains this leader sequence. We therefore examined if MD-2s exists as a stably secreted protein. Myc-tagged proteins were immunoprecipitated from culture supernatants collected from HEK293 cells transiently expressing MD-2s-Myc. As shown in Fig. 2e lane 2, a secreted product corresponding to MD-2s was detected.
We investigated the requirement for MD-2s in LPS mediated NF-κB activation. It is known that the soluble form of full length MD-2 confers LPS responsiveness to cells expressing TLR4 (26, 28, 29), we therefore investigated if soluble MD-2s was also bioactive. Culture supernatants were collected from HEK293 cells transiently expressing control vector, MD-2, or MD-2s, and incubated with HEK293 cells stably transfected with TLR4 and an NF-κB-dependent luciferase reporter gene. Consistent with published results, soluble MD-2 conferred the ability of these reporter cells to respond to LPS by inducing both NF-κB activation and IL-8 secretion (Fig 3a and 3b, respectively). In stark contrast the secreted form of MD-2s could not activate either of these responses (Fig. 3a and 3b). We next assessed the capacity of MD-2s to mediate signal transduction triggered by LPS following transient transfection of the MD-2s expression vector. HEK293TLR4 cells stably expressing the NF-κB-dependent luciferase reporter gene were transiently transfected with plasmids encoding CD14 in combination with either MD-2 or MD-2s, and subsequently stimulated with LPS. Although full length MD-2 activated NF-κB and lead to secretion of IL-8 in the presence of LPS, overexpressed MD-2s remained inactive, even at higher concentrations (Fig. 3c and 3d). Taken together, these results suggest that it is essential for MD-2 to contain the region encoded by exon 2 in order to mediate LPS-induced TLR4 signaling.
Although MD-2 lacks transmembrane and intracellular regions, it can be membrane-bound through its association with the extracellular portion of TLR4 (30). Given that MD-2s failed to mediate LPS signaling, we next asked whether this might be due to an inherent inability to interact with TLR4. To address this possibility, we first investigated if MD-2s was located on the surface of TLR4 expressing cells. We transiently transfected TLR4-mCitrine expressing cells with plasmids encoding either MD-2 or MD-2s and performed flow cytometric analysis. We observed that MD-2s was bound to the cell surface of TLR4 expressing cells (Fig. 4a). We then examined the cellular distribution of MD-2s with respect to TLR4 by confocal microscopy. We found that MD-2s exhibited the same cellular distribution as MD-2 and colocalized with TLR4 on the cell surface (Fig. 4b). We next assessed if MD-2s was anchored to the membrane via association with TLR4. We observed that MD-2s immunoprecipitated together with TLR4 (Fig. 4c, lane 4). Furthermore, as has been reported with MD-2, both the non-glycosylated and glycosylated form of MD-2s immunoprecipitated with TLR4. This indicates that although MD-2s failed to confer LPS responsiveness to cells in the absence of MD-2, this cannot be attributed to an inability to associate with TLR4.
Having determined that MD-2s cannot reconstitute LPS signaling in cells lacking MD-2, we next investigated the effect of soluble MD-2s on TLR4 activation. HEK293 cells stably expressing TLR4 and an NF-κB-dependent luciferase reporter gene were incubated with supernatants collected from HEK293 cells that contained a constant amount of MD-2 but increasing concentrations of MD-2s. We noted that soluble MD-2s blocked NF-κB activation in response to LPS in a dose-dependent manner (Fig. 5a). We also observed that overexpressed MD-2s inhibited LPS-induced IL-8 secretion (Fig. 5b). These findings indicate that MD-2s functions as a negative regulatory protein in the TLR4 signaling pathway, suggesting that MD-2s may be used as a therapeutic or preventative agent for modulating an overactivated MD-2-TLR4 immune response.
We next analyzed the published structural models of MD-2 to ascertain the structural effect of deleting exon 2. MD-2 consists of a β-cup fold with two anti-parallel β-sheets, one composed of six β-strands (numbered 1/2/9/8/5/6) and the other of three (numbered 3/4/7) (31, 32). The missing exon 2 of MD-2s encodes the first two β-strands of the three-stranded β-sheet (β3 and β4 strands) (Fig. 5c). The hinges connecting β-strands 2 to 3 and 4 to 5 are also partially lost. Furthermore, the disulphide bond between Cys25 and Cys51, which assists in closing the MD-2 cavity and stabilizing the cup-like structure, is disrupted. The β6 and β7 strands that line the entrance to the deep hydrophobic cavity are still encoded by the mRNA of MD-2s.
Elucidation of the TLR4-MD-2-LPS complex revealed that LPS binding instigates the formation of a receptor multimer which is composed of two copies of the complex arranged symmetrically (14). Eleven residues are involved in the main dimerization interface of the TLR4-MD-2-LPS complex, of these 10 are still present in MD-2s. However, deletion of exon 2 in MD-2 implied that the ligand-binding pocket of MD-2s may be severely disrupted, suggesting that it may be unable to bind LPS efficiently. To address this possibility, HEK293 cells were transiently transfected with plasmids encoding either MD-2-Myc or MD-2s-Myc. Culture supernatants were then incubated with biotinylated-LPS and Myc-tagged proteins were immunoprecipitated. In agreement with previous studies (33–35), secreted MD-2 bound readily to LPS (Fig. 5d, lane 4). Interestingly, under similar conditions, an interaction between secreted MD-2s and LPS was also detected (Fig. 5d, lane 6), suggesting that soluble MD-2s may sequester LPS from binding to the MD-2-TLR4 complex and thus diminish TLR4 signal transduction.
Given that MD-2s inhibits LPS-induced TLR4 signaling, but still interacts with LPS and TLR4, we hypothesized that it may compete with MD-2 to form an interaction with TLR4. To investigate this possibility, we increased the expression level of MD-2s-Myc, but kept the concentration of the constructs for HA-MD-2 and Flag-TLR4 constant. As expected, both the glycosylated and non-glycosylated forms of MD-2 immunoprecipitated with TLR4 in HEK293 cells transiently transfected with plasmids encoding both proteins (Fig. 5e, lane 2). However, overexpression of MD-2s competitively inhibited binding of MD-2 to TLR4 (Fig. 5e, compare lanes 3 and 4 to lane 2), indicating that MD-2s downregulates TLR4 signaling by inhibiting the formation of an active MD-2-TLR4 signaling complex.
An inappropriately mounted or dysregulated immune response can cause considerable morbidity and mortality in a number of diseases. One such example is sepsis, which is among the most common causes of death in the United States, with over 750,000 cases presenting annually, of which more than one-quarter are fatal (36). Excessive inflammation is the hallmark of a number of related infectious pathologies as well, including sepsis, acute respiratory distress syndrome (ARDS) and multiple organ failure (37). LPS derived from bacterial sources can contribute to these diseases, and does so by interacting with MD-2 and TLR4. To circumvent an overactivated host immune response to LPS, it is imperative that TLR4 signal transduction be tightly regulated, but the precise molecular mechanisms by which this is accomplished are only partly understood.
Here we further elucidate the complexities involved in averting a prolonged and dysregulated immune response to LPS by the identification of a naturally occurring alternatively spliced isoform of human MD-2, which we have termed MD-2s. We report that human MD-2s is generated by skipping exon 2 of the MD-2 gene, which leads to an in-frame deletion of 30 amino acids spanning positions 39–69, and one amino acid substitution (D38G). Under similar conditions and using primers specific to the murine MD-2 gene, we could not detect a corresponding murine splice variant. A prior study reported an alternatively spliced version of murine MD2 (MD-2B) (21), which lacks the first 54 bases of exon 3 and downregulates LPS signaling. However, there are no data provided as to whether it is secreted or inducible and the human relevance is unknown (see Table II for comparison between MD-2s and MD-2B).
Our results identify MD-2s as an important negative regulatory component of the TLR4 signaling pathway. The mRNA expression profile of MD-2s in human tissues revealed that it is ubiquitously expressed, suggesting that this isoform may perform a widespread role in modulating TLR4-mediated responses. Previous studies have shown that IFN-γ exerts anti-inflammatory responses by inducing specific secreted inhibitors, examples include the IL-1 receptor antagonist (IL-1Ra) (38) and IL-18 binding protein (IL-18BP) (39). Both molecules suppress the activity of IL-1 and IL-18 respectively. Many LPS-inducible negative regulators have also been identified, such as smTLR4 and MyD88s. We determined that MD-2s is upregulated in response to IFN-γ, IL-6 and LPS, indicating that MD-2s may be a key component involved in the negative regulation of TLR4 signaling.
Similar to full-length MD-2, MD-2s is a secreted glycoprotein. However, MD-2s overexpression failed to trigger NF-κB activation and IL-8 secretion following LPS treatment, indicating the importance of exon 2 for MD-2 function. Importantly, we also observed that MD-2s negatively regulated both NF-κB activation and IL-8 secretion following LPS stimulation, suggesting that MD-2s may be used as a therapeutic or preventative agent for modulating endotoxemia. Whereas the murine isoform, MD-2B, inhibited TLR4 from being expressed on the cell surface (21), MD-2s is anchored to the cell surface of TLR4 expressing cells, and both proteins localize together on the cell membrane. We also determined that MD-2s and TLR4 immunoprecipitated together. This may have been predicted, given that MD-2s retains most of the residues reported to be essential in mediating a MD-2-TLR4 interaction, with the exception of I66 and R68 (31, 40). In addition, MD-2s immunoprecipitated with LPS. This is consistent with a study demonstrating that a 15 residue peptide fragment of MD-2, encompassing the F126 loop (positions 119–132), still binds LPS (41). Additional studies have verified that residues within this fragment of MD-2 are essential for LPS binding (34, 40, 42). This region is preserved in MD-2s and most likely confers the ability of MD-2s to associate with LPS. Furthermore, although the hydrophobic pocket of MD2-s is predicted to be disrupted, with the exception of K58 all the MD-2 residues that have been shown to be involved in the main dimerization interface of the TLR4-MD-2-LPS complex, are preserved in MD-2s. Whilst it appears that these residues are sufficient to form an effective interaction between MD-2s and LPS, the binding affinity is most likely affected.
Several studies have shown that negative regulators can control TLR signal transduction by inhibiting the formation of active signaling complexes, including the recently identified splice variant TAG (20), IRF-4 (43), RP105 (44) and the IL-1Ra (45–47). Based on our results, we propose that MD-2s functionally modulates TLR4 signaling by inhibiting the formation of an active MD-2-TLR4 signaling complex. In addition, it is possible that MD-2s may behave like a decoy co-receptor by binding LPS and TLR4 to form a non-functional complex that does not activate NF-κB, thereby negatively regulating signaling.
Collectively our results define an important mechanistic role for MD-2s in modulating the LPS-TLR4 signal transduction pathway at the initial phase of activation. MD-2s therefore represents a prospective target for pharmacological intervention and development of new therapeutic and preventive strategies for sepsis and other diseases resulting from an over-exuberant MD2-TLR4-induced immune response.
We sincerely thank P. Sun for technical assistance; K. Miyake (Tokyo University) for the plasmid encoding Flag MD-2; F.J. Candal (Center for Disease Control and Prevention) for the HMECs, and Terence M. Doherty (Cedars-Sinai Medical Center) for editorial assistance.
Supported by the National Institutes of Health Grants (HL-66436 and AI-058128 to MA).