Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus caseiinhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells
© Hiramatsu et al.; licensee BioMed Central Ltd. 2014
Received: 1 October 2013
Accepted: 1 May 2014
Published: 10 May 2014
Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs.
We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability.
The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.
KeywordsCaspase-1 Cholera toxin B subunit GM1 ganglioside Interleukin-1β Lactobacillus casei YVAD
Lactic acid bacteria (LAB) are not pathogenic and their classification is “generally recognized as safe”. Over the past several decades, LAB have been used in foods and medicines because they confer beneficial effects on the health of the host. Moreover, after their administration, LAB are delivered live to the intestine, colonizing the mucosal surface and exerting various effects . Therefore, LAB that produce heterologous proteins have been used as bacterial vectors for the delivery of functional proteins to the intestine. Many studies using recombinant DNA technology have used Lactobacillus species, which are present in large numbers in the human gut and are resistant to gastric and bile acids . These live recombinant lactobacilli colonize the intestinal mucosal surface and produce the desired protein . Although Escherichia coli has generally been used for the production of heterologous proteins, coliform lipopolysaccharide (LPS) contamination always poses a problem. In contrast to E. coli, Lactobacillus species are gram-positive bacteria and consequently do not contain LPS. Therefore, we selected Lactobacillus species for the secretion of functional heterologous proteins.
Cholera toxin (CT) is an enterotoxin produced by Vibrio cholerae, which is composed of a toxic A subunit (CTA) and nontoxic B subunit (CTB). CT gains entry to intestinal epithelial cells (IECs) when CTB binds to GM1 ganglioside, a cell-surface receptor present on mammalian cells. CTB alone can translocate into IECs through the GM1 ganglioside without toxicity . Many groups have reported that recombinant CTB (rCTB) expressed in various bacteria, yeasts, and plants also binds to GM1 ganglioside. Previously, we constructed a recombinant Lactobacillus casei that secretes CTB, and showed that the rCTB secreted by L. casei has GM1-ganglioside-binding activity similar to that of CT from V. cholerae. Recombinant fusion proteins of CTB with functional proteins and peptides, such as vaccine antigens  and the insulin B chain peptide , have been used as carriers to deliver these proteins and peptides to IECs, because they also bind to GM1 ganglioside. However, it has not been determined whether recombinant fusion proteins of CTB with functional proteins or peptides expressed by Lactobacillus species bind GM1 ganglioside and translocate into IECs.
The synthetic tetrapeptide composed of tyrosine, valine, alanine, and aspartic acid (YVAD) is a specific inhibitor of caspase-1 . Caspase-1 catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its precursor (pro-IL-1β), and its overexpression in and secretion from IECs exacerbates intestinal inflammation [9, 10]. Caspase-1 is also produced as an inactive precursor, pro-caspase-1, which is activated by inflammatory stimuli, such as LPS and mature caspase-1 itself [11, 12]. Therefore, YVAD has anti-inflammatory properties, acting as a decoy substrate for caspase-1 instead of pro-IL-1β and pro-caspase-1. However, recombinant bacteria expressing or secreting YVAD have not been reported because it is difficult to express and secrete recombinant low-molecular-weight peptides in bacteria. Furthermore, for YVAD to inhibit caspase-1 activation and subsequent IL-1β secretion, it must be translocated into IECs. However, the cell permeation capacity of YVAD is low because of its strong polarity . Here, we investigated whether fusing rCTB to YVAD would allow the secretion of recombinant YVAD from L. casei and facilitate the translocation of YVAD into IECs.
In this study, we constructed L. casei that secretes a recombinant fusion protein of CTB with YVAD (rCTB–YVAD) and confirmed that rCTB–YVAD secreted by L. casei binds to GM1 ganglioside, translocates into human epithelial colorectal adenocarcinoma Caco-2 cells used as a model of IECs, and inhibits the activation of caspase-1 and subsequent IL-1β secretion from Caco-2 cells.
Results and discussion
Secretion of rCTB-YVAD by L. caseitransformed with pSCTB-YVAD
Purification of rCTB–YVAD secreted by pSCTB–YVAD-transformed L. casei
Viability of Caco-2 cells after rCTB–YVAD and rCTB treatment
Translocation of rCTB-YVAD and rCTB into Caco-2 cells
The cell permeation of functional peptides, such as vaccine antigens and the insulin B chain peptide, is increased by their fusion with CTB [6, 7], because CTB translocates easily into IECs by binding to GM1 ganglioside . For YVAD to inhibit caspase-1 activation and subsequent IL-1β secretion, it is necessary for YVAD to translocate into Caco-2 cells. Our results suggest that the fusion of CTB to YVAD contributed to the translocation of YVAD into Caco-2 cells. The translocation of rCTB–YVAD into the cells, regardless of the presence or absence of LPS, suggests that rCTB–YVAD translocates into Caco-2 cells through GM1 ganglioside, which is constantly expressed, regardless of the inflammatory status.
Inhibitory effect of rCTB–YVAD on LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells
The expression and secretion of YVAD by bacteria have been limited by its low molecular weight. Therefore, we constructed recombinant L. casei secreting YVAD as a fusion protein with CTB and showed that YVAD secreted by L. casei inhibits caspase-1 activation and subsequent IL-1β secretion. The results of this study indicate that YVAD secreted by bacteria exerts an anti-inflammatory effect. Meng et al. also reported that recombinant green fluorescent protein (GFP) fused to the C-terminus of CTB expressed in silkworms emitted green fluorescence similar to that emitted by recombinant GFP alone . That report and the results of the present study suggest that the effects of functional peptides or proteins are not abolished by their fusion to the C-terminus of CTB.
We constructed an L. casei that secretes a recombinant CTB protein fused to YVAD. Although rCTB–YVAD bound GM1 ganglioside and translocated into Caco-2 cells, like rCTB, rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion without affecting cell viability. These results indicate not only that a recombinant fusion protein of CTB with a functional peptide secreted by L. casei has GM1-ganglioside-binding activity, but also that recombinant YVAD secreted by L. casei exerts an anti-inflammatory effect. The results of this study suggest that rCTB secreted by L. casei has potential utility as a system for the delivery of YVAD into IECs. However, we were unable to examine the anti-inflammatory effects of rCTB–YVAD-secreting L. casei in this study because L. casei could not grow or secrete rCTB–YVAD in MEM. We confirmed that it is difficult to completely mimic the intestinal environment in in vitro experiments. Therefore, further studies, such as an in vivo study of the ingestion of rCTB–YVAD secreting L. casei, are required to examine the effects of rCTB–YVAD-secreting L. casei. If successful, such a study would confirm that rCTB-secreting L. casei has potential utility as a delivery system for functional peptides into the intestine.
Bacterial strains and culture conditions
The strains used in this study were L. casei ATCC 27092 and E. coli DH5α. L. casei was grown at 37°C in MRS broth to produce the recombinant strain or at 30°C in MRS/K (MRS with 0.2 M potassium phosphate buffer) to produce the cell culture supernatant . Erythromycin (5 μg/ml) was added to MRS or MRS/K to select the recombinant strain. E. coli was grown at 37°C in LB medium with or without ampicillin (100 μg/ml).
Construction of the rCTB–YVAD secretion vector
PCR was performed using the plasmid pSCTB  as the template DNA, KOD-Plus- DNA polymerase (Toyobo, Osaka, Japan), and primers containing the YVAD-coding sequence fused to the C-terminus of the CTB gene: sense, 5′-caccaccaccaccaccactaaaggccttc-3′; anti-sense, 5′-atcagcaacataatttgccatactaattgc-3′. Initial denaturation was at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec, and extension at 68°C for 7 min. The DNA fragment of about 6,600 bp was separated by agarose gel electrophoresis and extracted from the gel with the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The extracted DNA was phosphorylated at the 5′ end with T4 polynucleotide kinase (TaKaRa Bio, Shiga, Japan) and self-ligated with T4 DNA ligase (TaKaRa Bio). The rCTB–YVAD secretion vector pSCTB–YVAD was confirmed by sequencing, and then introduced into L. casei by electroporation, as described previously .
Secretion of rCTB–YVAD by L. casei
Overnight cultures of L. casei transformed with pSCTB–YVAD or pSCTB were inoculated into MRS/K medium to an optical density at 600 nm (OD600) of 0.05, and the cells were grown at 30°C until they reached an OD600 of 2.0. The culture supernatants were collected by centrifugation (7,000 × g, 5 min, 4°C) and concentrated 10-fold with Amicon Ultra Centrifugal Filter Units (10 kDa; Merck Millipore, Tokyo, Japan). The secretion and specific GM1-ganglioside-binding activities of rCTB–YVAD and rCTB in the concentrated supernatant were confirmed with immunoblotting using an antibody directed against CT or a GM1 enzyme-linked immunosorbent assay (GM1-ELISA), respectively.
The concentrated supernatants of L. casei (20 μl/lane), rCTB–YVAD (100 ng/lane), and cellular protein extracts of Caco-2 cells (50 μg/lane) were subjected to immunoblotting analysis. They were separated with SDS-PAGE (10–18%) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, UK). Antibodies directed against CT (Sigma-Aldrich, St. Louis, MO) and β-actin (Cell Signaling Technology, Boston, MA) were used as the primary antibodies. Alkaline phosphatase (AP)-labeled anti-rabbit IgG antibody (Cell Signaling Technology) was used as the secondary antibody, and binding was detected with a chemiluminescent substrate of AP (CDP-Star Reagent; Biolabs, Beverly, MA).
A GM1-ELISA was performed to determine specific GM1-ganglioside-binding activities. Briefly, 96-well microtiter plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were coated with 5 μg/ml monosialoganglioside GM1 (Sigma-Aldrich) diluted in bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), and incubated overnight at 4°C. After incubation, the plates were washed three times with PBS supplemented with 0.05% Tween-20 (PBS-T), and then blocked with PBS containing 1% bovine serum albumin (Nacalai Tesque, Kyoto, Japan) at 37°C for 2 h. After the plates were washed, the concentrated supernatant of L. casei (100 μl/well), rCTB–YVAD (50 ng/well), or rCTB (50 ng/well) was applied to the wells and incubated at 37°C for 2 h. The plates were incubated at 37°C for 2 h with antibody directed against CT and AP-labeled anti-rabbit IgG antibody used as the primary and secondary antibodies, respectively. The plates were incubated with AP substrate (Sigma-Aldrich) at 37°C for 20 min, and the OD405 was then measured with a microplate reader (ImmunoMini Nj-2300; Nunc, Rochester, NY).
Purification of rCTB–YVAD secreted by L. casei
rCTB–YVAD from the culture supernatant of L. casei transformed with pSCTB–YVAD was purified using the His-tag and an affinity resin containing bound nickel ions. The culture supernatant of L. casei transformed with pSCTB–YVAD was collected by centrifugation (12,000 × g, 30 min, 4°C) after growth in MRS/K medium at 30°C until the OD600 was 2.0. After the supernatant was filtered at 0.22 μm, imidazole was added to a final concentration of 20 mM, and the culture supernatant was then adjusted to pH 7.0. Nickel resin (Ni Sepharose High Performance; GE Healthcare) was added to the culture supernatant and then mixed gently overnight at 4°C. The open column was filled with resin, and then washed with wash buffer (10 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). rCTB–YVAD bound with nickel resin was eluted with elution buffer (10 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4). The eluted rCTB–YVAD was concentrated and the buffer replaced with PBS using Amicon Ultra Centrifugal Filter Units (10 kDa). The protein concentration of rCTB–YVAD was determined with Coomassie Protein Assay Reagent (Pierce, Perbio Science, Bonn, Germany). The purity of rCTB–YVAD was confirmed with SDS-PAGE on 17% polyacrylamide gel, followed by CBB staining and immunoblotting using an antibody directed against CT. The GM1-ganglioside-binding activities of purified rCTB–YVAD and rCTB were confirmed with GM1-ELISA.
Caco-2 cell viability assay
Caco-2 cells were cultured as described previously . Aliquots of 5 × 103 Caco-2 cells were plated in each well of a 96-well plate (Nunc). The cells were treated with three concentrations of rCTB–YVAD or rCTB (10, 20, or 50 μM) in the presence of 10 μg/ml LPS from E. coli O55:B5 (Sigma-Aldrich). After incubation for 48 h, 20 μl of WST-1 Cell Proliferation Reagent (TaKaRa Bio) was added to each well. After 2 h, the OD450 and OD630 were measured with a microplate reader. Cell viability was calculated as (OD450 – OD630 of treated cells/OD450 – OD630 of untreated control cells) × 100%.
Detection of translocated rCTB–YVAD and rCTB in Caco-2 cells
Aliquots of 7 × 105 Caco-2 cells were plated in each well of six-well plates (Nunc). Cells were treated with 50 μM rCTB–YVAD or rCTB in the absence or presence of 10 μg/ml LPS. After incubation for 6 h, cellular protein extracts were prepared with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Kyungki-Do, South Korea), according to the manufacturer’s protocol. The protein concentrations of the cellular protein extracts were determined with Coomassie Protein Assay Reagent. Intracellular rCTB–YVAD and rCTB were detected by immunoblotting with an antibody directed against CT. Equal loading was confirmed with an antibody directed against β-actin.
Inhibitory effect on caspase-1 activity
Caspase-1 activity was determined with a modification of a previously described method [20, 21]. Aliquots of 1 × 107 Caco-2 cells were plated in 90 mm plastic culture dishes (Nunc) and treated with or without 10 μg/ml LPS for 12 h. The cells were washed with PBS and resuspended in buffer W (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, pH 7.4) supplemented with 10 mM DTT and 1 mM phenylmethylsulfonyl fluoride. The cells were incubated at 4°C for 15 min and disrupted by 20 passages through a 23G needle. The lysates were then centrifuged at 12,000 × g for 5 min at 4°C and the supernatants collected. The protein concentrations of the lysates were determined with Coomassie Protein Assay Reagent. Aliquots of 10 μg/μl lysate were incubated at 30°C for 2 h in the absence or presence of 50 μM rCTB–YVAD or rCTB. The caspase-1 activity in the 10-fold-diluted lysate was determined with a Caspase 1 Assay Kit, Colorimetric (Calbiochem, La Jolla, CA).
Measurement of IL-1β by ELISA
Aliquots of 7 × 105 Caco-2 cells were plated in each well of six-well plates. The cells were treated with 50 μM rCTB–YVAD and rCTB in the presence of 10 μg/ml LPS for 48 h. The cell supernatants were centrifuged at 15,000 × g for 5 min at 4°C and stored at −80°C until IL-1β analysis. The concentrations of IL-1β in the cell supernatants were determined with a human IL-1β ELISA Kit (R&D Systems, Abingdon, UK).
Data are presented as means ± SEM. Statistical analyses were performed with Origin Pro 8.1 (OriginLab, Northampton, MA). Differences were analyzed with one-way ANOVA followed by Tukey’s test. In all analyses, P < 0.05 was deemed to indicate significance.
Serum albumin binding region
Coomassie Brilliant Blue
Cholera toxin A subunit
Cholera toxin B subunit
Green fluorescent protein
GM1 enzyme-linked immunosorbent assay
Intestinal epithelial cells
Lactic acid bacteria
Precursor of caspase-1
Precursor of IL-1β
Recombinant fusion protein of CTB with YVAD
Secretory signal sequence
Tetrapeptide composed of tyrosine, valine, alanine, and aspartic acid.
This research was supported by the following grants: JSPS KAKENHI (grant numbers 24658099 and 24659060); JST Project to Develop Innovative Seeds (grant number 12–046); and the Central Research Institute from Fukuoka University (grant numbers 126012 and 127007).
- del Rio B, Dattwyler RJ, Aroso M, Neves V, Meirelles L, Seegers JF, Gomes-Solecki M: Oral immunization with recombinant Lactobacillus plantarum induced a protective immune response in mice with Lyme disease. Clin Vaccine Immunol. 2008, 15: 1429-1435. 10.1128/CVI.00169-08.View ArticleGoogle Scholar
- Jung SW, Kim WJ, Lee KG, Kim CW, Noh WS: Fermentation characteristics of exopolysaccharide-producing lactic acid bacteria from sourdough and assessment of the isolates for industrial potential. J Microbiol Biotechnol. 2008, 18: 1266-1273.Google Scholar
- Pouwels PH, Lee RJ, Shaw M, den Bak-Glashouwer MJ H, Tielen FD, Smit E, Martinez B, Jore J, Conway PL: Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes. Int J Food Microbiol. 1998, 41: 155-167. 10.1016/S0168-1605(98)00048-8.View ArticleGoogle Scholar
- Fishman PH: Role of membrane ganglioside in the binding and action of bacterial toxins. J Membr Biol. 1982, 69: 85-97. 10.1007/BF01872268.View ArticleGoogle Scholar
- Okuno T, Kashige N, Satho T, Irie K, Hiramatsu Y, Sharmin T, Fukmits Y, Uyeda S, Harakuni T, Miyata T, Arakawa T, Imoto M, Toda A, Nakashima Y, Miake F: Expression and secretion of cholera toxin B subunit in lactobacilli. Biol Pharm Bull. 2013, 36: 952-958. 10.1248/bpb.b12-01021.View ArticleGoogle Scholar
- Harakuni T, Sugawa H, Komesu A, Tadano M, Arakawa T: Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to taeget recombinant vaccine antigens to mucosal immune systems. Infect Immun. 2005, 73: 5654-5665. 10.1128/IAI.73.9.5654-5665.2005.View ArticleGoogle Scholar
- Yuki Y, Hara-Yakoyama C, Guadiz AA, Udaka S, Kiyono H, Chatterjee S: Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune disease. Biotechnol Bioeng. 2005, 92: 803-809. 10.1002/bit.20654.View ArticleGoogle Scholar
- Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA: Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998, 273: 32608-32613. 10.1074/jbc.273.49.32608.View ArticleGoogle Scholar
- Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J, Elliston KO, Ayala JM, Casano FJ, Chin J, Ding GJF, Egger LA, Gaffney EP, Limjuco G, Palyha OC, Raju SM, Rolando AM, Salley JP, Yamin TT, Lee TD, Shively JE, MacCross M, Mumford RA, Schmidt JA, Tocci MJ: A novel heterodimetric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992, 356: 768-774. 10.1038/356768a0.View ArticleGoogle Scholar
- Mahida YR, Wu K, Jewell DP: Enhanced production of interleukin 1-beta by mononuclear cells isolated from mucosa with active ulcerative colitis of Crohn’s disease. Gut. 1989, 30: 835-838. 10.1136/gut.30.6.835.View ArticleGoogle Scholar
- Schumann RR, Belka C, Reuter D, Lamping N, Kirschning CJ, Weber JR, Pfeil D: Lipopolysaccharide activates caspase-1 (interleukin-1-converting enzyme) in cultured monocytic and eondothelial cells. Blood. 1998, 91: 577-584.Google Scholar
- Ramage P, Cheneval D, Chvei M, Graff P, Hemmig R, Heng R, Kocher HP, Mackenzie A, Memmert K, Revesz L, Wishart W: Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor. J Biol Chem. 1995, 270: 9378-9383. 10.1074/jbc.270.16.9378.View ArticleGoogle Scholar
- Charrier JD, Durrant SJ, Studley J, Lawes L, Weber P: Synthesis and evauation of novel prodrugs of caspase inhibitors. Bioorg Med Chem Lett. 2012, 22: 485-488. 10.1016/j.bmcl.2011.10.102.View ArticleGoogle Scholar
- Sadeghi H, Bregenholt S, Wegmann D, Petersen JS, Holmgren J, Lebens M: Genetic fusion of human insulin B-chain to the B subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo. Immunology. 2002, 106: 237-245. 10.1046/j.1365-2567.2002.01413.x.View ArticleGoogle Scholar
- Kim TG, Kim HY, Yang MS: Cholera toxin B subunit-domain ΙΙΙ of dengue virus envelope glycoprotein E fusion protein production in transgenic plants. Protein Expr Purif. 2010, 74: 236-241. 10.1016/j.pep.2010.07.013.View ArticleGoogle Scholar
- Liljegvist S, Ståhl S, Andréoni C, Binz H, Uhlén M, Murby M: Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. J Immunol Methods. 1997, 210: 125-135. 10.1016/S0022-1759(97)00170-1.View ArticleGoogle Scholar
- Dertzbaugh MT, Cox LM: The affinity of cholera toxin for Ni2+ ion. Protein Eng. 1998, 11: 577-581. 10.1093/protein/11.7.577.View ArticleGoogle Scholar
- Meng Q, Wang W, Shi X, Jin Y, Zhang Y: Protection against autoimmune diabetes by silkworm-produced GFP-tagged CTB-insulin fusion protein. Clin Dev Immunol. 2011, 2011: 831704-View ArticleGoogle Scholar
- Hiramatsu Y, Satho T, Irie K, Shiimura S, Okuno T, Sharmin T, Uyeda S, Fukumitsu Y, Nakashima Y, Miake F, Kashige N: Differences in TLR9-dependent inhibitory effects of H2O2-induced IL-8 secretion and NF-kappa B/I kappa B-alpha system activation by genomic DNA from five Lactobacillus species. Microbes Infect. 2013, 15: 96-104. 10.1016/j.micinf.2012.11.003.View ArticleGoogle Scholar
- Martinon F, Burns K, Tschopp J: The inflammasome: a molecular platform triggering acivation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002, 10: 417-426. 10.1016/S1097-2765(02)00599-3.View ArticleGoogle Scholar
- Yamamoto M, Yaginuma K, Tsutsui H, Sagara J, Guan X, Seki E, Yasuda K, Yamamoto M, Akira S, Nakanishi K, Noda T, Taniguchi S: ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules. Genes Cells. 2004, 9: 1055-1067. 10.1111/j.1365-2443.2004.00789.x.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.