Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Surface-Displayed IL-10 by Recombinant Lactobacillus plantarum Reduces Th1 Responses of RAW264.7 Cells Stimulated with Poly(I:C) or LPS

  • Cai, Ruopeng (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Jiang, Yanlong (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Yang, Wei (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Yang, Wentao (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Shi, Shaohua (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Shi, Chunwei (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Hu, Jingtao (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Gu, Wei (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Ye, Liping (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Zhou, Fangyu (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Gong, Qinglong (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Han, Wenyu (College of Veterinary Medicine, Jilin University) ;
  • Yang, Guilian (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University) ;
  • Wang, Chunfeng (College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University)
  • Received : 2015.09.10
  • Accepted : 2015.11.24
  • Published : 2016.02.28
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Abstract

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surface-displayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

Keywords

Introduction

Poly-γ-glutamic acid synthetase A (pgsA) is a constituent protein of the polyglutamic acid (PGA) synthetase system of Bacillus subtilis, which can firmly anchor certain enzymatic systems on cell walls. Narita et al. [23] first developed a pgsA bacterial surface-displayed system, and enzymes from different sources were displayed successfully on the surface of Escherichia coli using this anchor motif. The system could be applied to gram-positive bacteria because of its high efficiency of surface display; however, to date, studies have been limited to Lactobacillus casei and Lactococcus lactis. Poo et al. [25] reported anchoring Human Papilloma Virus Type 16 (HPV-16) E7 protein to the surface of L. casei using this system prior to oral administration in C57BL/6 mice, and they found that the recombinant strain led to systemic and mucosal immune responses in addition to antitumor effects. Yoon et al. [31] displayed HPV-16 L2 protein on the surface of the same strain using a pgsA system, which showed that the recombinant strain could both mediate comprehensive immune responses and result in the cross-neutralization of other subtypes of papilloma viruses. Additionally, Lei et al. [18] also showed that ferrets that were administered L. lactis-pgsA-HA1 intranasally could robustly develop humoral and mucosal immune responses, as well as notable HI titers. Importantly, treated ferrets were completely protected from H5N1 virus challenge. Lei et al. [19] also revealed that mice vaccinated orally with L. lactis-pgsA-NA could elicit immune protection against a range of influenza A viruses.

Lactobacillus plantarum is both a common lactic acid bacteria found in many food products and a natural inhabitant of the human gastrointestinal tract (GIT). It has a wide range of applications in industrial lactic acid fermentation and healthcare. Similar to a variety of beneficial lactic acid bacteria, this strain can suppress colonization by exogenous bacteria via the secretion of lactic acid, bacteriocin, and other factors, which promotes a stable biological barrier by maintaining the internal relations of intestinal microbiota, which stabilizes the intestinal microecology. Additionally, L. plantarum can inhibit pathogen invasion and colonization by utilizing essential growth substances of exogenous strains and preventing their adhesion to the GIT. Moreover, probiotics made by L. plantarum can enhance systemic immunity. Indeed, Kotzamanidis et al. [15] found that this bacteria can effectively prevent the occurrence of some diseases as a consequence of specific characteristics (e.g., hydrophobicity and high adhesion) of the surface of this strain that have potential immunomodulatory properties. For example, L. plantarum 2035 can enhance neutrophil chemotaxis and phagocytosis. Moreover, the activation of PRRs and secretion of multiple cytokines can also be mediated by this bacteria. However, these cytokines are predominantly of the Th1-type. Kim et al. [13] found that levels of IL-23 p19 in THP-1 cells stimulated by lipoteichoic acids from this strain could suppress the effects of IL-10. Maeda et al. [21] established that the heat-killed strain could evoke a small amount of Th1-type cytokines. Therefore, L. plantarum can be harnessed to treat Th2-type allergic diseases by bringing the Th1/Th2 balance to a normal level [17]. Additionally, the preventive and therapeutic effects on chronic digestive diseases, cardiovascular disease, and viral infections by the strain were also confirmed [3,12,27].

Because of its outstanding biological activities and targeting effects in vivo, L. plantarum has become utilized as a delivery system for exogenous proteins. The hydrolysis in the GIT has been largely overcome by utilizing this strain as a vehicle for delivering oral pharmaceutical proteins, but the products encoded by plasmids are mostly localized in the cytoplasm [4]. The cell wall of L. plantarum remains the biggest obstacle for achieving extracellular expression of a foreign gene, although it is thinner than that of other species of lactic acid bacteria. Additionally, severe metabolic burdens to L. plantarum can be caused by some toxic substances from exogenous proteins in the cytoplasm, so the functions of recombinant strains can often be impaired.

As an ideal type of anchorin, pgsA has been established as a foundation to solve the extracellular transport problem of heterogenous proteins expressed by L. plantarum. In our present study, the pgsA surface-displayed system was used to achieve the extracellular transport of exogenous proteins in recombinant L. plantarum. Surface-displayed pgsA was verified by many tests, but we found that expression was negatively affected by its long gene sequence (1,143 bp). Therefore, pgsA’, a shortened anchored gene, was obtained using computational predictions of the surface probability, antigenic index, and hydrophilicity of pgsA with DNASTAR software (DNASTAR Software, WI, USA). Then, murine IL-10 was used as an exogenous protein for recombinant L. plantarum with or without anchored fragments, and the surface expression of this protein on bacteria was measured by western blot assay. As Th1-type cytokines expressed by RAW264.7 cells can indicate an inflammatory response induced by irritants, we investigated whether IL-10 expressed by recombinant L. plantarum could specifically modulate cytokine secretion. Cells were co-cultured with recombinant L. plantarum before stimulation with Poly(I:C), a TLR3 ligand, or lipopolysaccharide (LPS), a TLR4 ligand. Then, the expression of pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, IL-6, and nuclear transcription factor NF-κB p65, were measured by ELISA, qRT-PCR, and immunofluorescence analyses.

 

Materials and Methods

Bacteria and Plasmid Vectors

The Escherichia coli-Lactobacillus shuttle vector pSIP409 and its host strain L. plantarum NC8 were generously provided by Professor A. Kolandaswamy (Madurai Kamaraj University, India). L. plantarum NC8 was cultivated in DeMan-Rogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, UK) at 30℃ without agitation. Trans10 chemically competent cells (TransGen, Beijing, China) were purchased as a cloning host and were cultured in Luria-Bertani (LB) medium at 37℃ with shaking at 180 rpm. Solid media required additional supplementation with 1.5% agar. Unless otherwise specified, antibiotic concentrations were 200 μg/ml ampicillin for recombinant E. coli, and 250 or 5 μg/ml erythromycin for recombinant E. coli or Lactobacillus, respectively.

pgsA (1,151 bp) with restriction sites NcoI and XbaI, as well as IL-10-1 (550 bp) with restriction sites XbaI and HindIII were sequence-optimized before synthesis by Generay Biotech Co., Ltd (Shanghai, China). Plasmid-specific primers were also synthesized and are presented in Table 1. pgsA’ and IL-10-2 with NcoI and XbaI restriction sites were amplified from the synthetic genes. All gene fragments were ligated with pMD 18-T Simple Vector and digested with corresponding restriction enzymes (all from Takara Biotechnology (Dalian) Co., Ltd, China). Then, enzyme-digested products were ligated to pSIP409 in the correct sequence (Fig. 1B). Plasmids were isolated using an E.Z.N.A. Plasmid Mini Kit, while gene fragments and linearized plasmids were purified with an E.Z.N.A. Gel Extraction Kit (all from Omega Bio-Tek Inc., Doraville, GA, USA). Amplified fragments from the plasmid vectors listed above were confirmed based on DNA sequencing carried out by Sangon Biotech Co., Ltd (Shanghai, China). Lastly, recombinant pSIP409 plasmids were transformed by electroporation once the L. plantarum NC8 competent cells were prepared as previously described [14]. Subsequently, L. plantarum-IL-10, L. plantarum-pgsA-IL-10, and L. plantarum-pgsA’-IL-10 were obtained, while L. plantarum-pgsA was constructed as a control.

Table 1.Sequences of PCR primer sets.

Fig. 1.Surface expression of IL-10 on Lactobacillus plantarum. (A) To identify the truncation sites, related indicators of the surface expression of pgsA, such as surface probability (Yellow), antigenic index (Pink), and hydrophilicity (Blue) were predicted using DNASTAR Protean software. (B) pSIP409-pgsA (pgsA’) plasmid vectors were constructed for IL-10 expression on the surface of Lactobacillus plantarum. (C) Equal protein loading was confirmed by SDS-PAGE. (D) Western blot analysis demonstrated the surface expression of IL-10 on L. plantarum with anti-IL-10 monoclonal antibody and HRP-conjugated goat anti-rat IgG antibody. Lane 1, L. plantarum-IL-10; Lane 2, L. plantarum-pgsA-IL-10; Lane 3, L. plantarum-pgsA’-IL-10. The 17, 62, and 41 kDa protein bands correspond to the anticipated sizes of IL-10 protein, and pgsA-IL-10 and pgsA’-IL-10 fusion proteins, respectively. (E) Protein grey value analysis of IL-10.

Cell Culture

The RAW264.7 cell line (ATCC HTB37) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum that contained 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco BRL, MD, USA) at 37℃ with 5% CO2. Cells were grown to 70–90% confluency on the day of transfection and were subjected to no more than 20 passages to prevent effects introduced by aging and insensitivity to irritants.

When adherent cells reached a density of 1 × 106 cells/ml, 5 × 106 CFU/ml recombinant L. plantarum was added to the supernatant (without antibiotics) to be co-incubated with cells for 4 h at 37℃; phosphate-buffered saline (PBS) was added to the control group. Subsequently, 100 μg/ml Poly(I:C) (P0913; Sigma) or 100 ng/ml LPS (L2880; Sigma) were added to stimulate the cells for 4 or 12 h, respectively.

Western Blot Analysis

Recombinant L. plantarum cells were cultured in MRS broth supplemented with erythromycin. Sakacin P (SppIP) was added to the culture medium at OD600nm ≈ 0.3 to induce the expression of heterologous proteins by plasmids. After induction at 30℃ for 8 h, strains were harvested, pelleted, and washed three times with 500 μl of PBS, resuspended in 500 μl of PBS, and handled with the method of freezing-thawing as previously described [5]. Samples were placed on ice and quantified using a BCA kit (KeyGEN, Nanjing, China). Then, 120 μl of cell lysate was mixed with 40 μl of 4× Protein SDS PAGE Loading Buffer (Takara, Dalian, China) and boiled for 10 min. Samples were centrifuged at 12,000 rpm for 2 min at 4℃, and equal amounts of supernatants were assessed by 12% SDS–PAGE and transferred to PVDF membranes (Millipore, USA). Mouse IL-10 was detected using an anti-IL-10 monoclonal antibody (ab33471; Abcam, USA) and HRP-conjugated goat anti-rat IgG antibody (A21040; Abbkine, USA). Both antibodies were diluted in 5% nonfat dried milk (BD, USA) in TBST. Blots were developed with Luminata Classico Western HRP Substrate (Millipore, USA) and measured using a chemiluminescence imaging system (Tanon 5200, Shanghai, China).

Measurements of Cytokine Concentrations

RAW264.7 cells were re-plated in 12-well plates (NEST, Wuxi, China) and treated as described above. Supernatants were harvested and centrifuged, and then stored at -80℃ before use. Cells were stored and later prepared for qRT-PCR. Concentrations of IL-1β, IL-6, TNF-α, and IFN-γ in supernatants were measured using ELISA kits (Excell, Shanghai, China) according to the manufacturer’s instructions. Results were determined using a microplate reader at OD450nm (Bio-Rad, USA).

Quantitative Analyses of mRNA Expression

The cells mentioned above were washed with PBS and total RNA was extracted using the MiniBEST Universal RNA Extraction Kit. RNA quality was confirmed using an ultramicro spectrophotometer (Thermo Scientific, USA), and less than 1 μg of total RNA was converted to cDNA using the PrimeScript RT reagent Kit with gDNA Eraser. Subsequently, qRT-PCR for murine IL-1β and IL-6 was performed utilizing SYBR Premix Ex Taq (TliRNaseH Plus; all from Takara, Dalian, China). Primers were designed and synthesized by Takara Biotechnology (Dalian) Co., Ltd (Table 1). Two-step programs were run on a Mastercycler ep realplex (Eppendorf, Germany) and all reactions were run in triplicate. Reactions were carried out as follows: initial denaturation at 95℃ for 30 sec, followed by 40 cycles of 5 sec at 95℃ and 30 sec at 60℃. Subsequently, the temperature was increased from 55℃ to 95℃ at a rate of 0.2℃/sec to establish a melting curve. Gene expression levels were calculated by the delta CT method as follows: fold-change = 2−ΔΔCt, where ΔCt = Ct (specific transcript) − Ct (housekeeping transcript) and ΔΔCt = ΔCt (treatment) − ΔCt (control).

Immunofluorescence Microscopy

RAW264.7 cells were re-plated in 35 mm glass-bottom dishes (NEST, Wuxi, China) and were treated as described above, and then were washed with PBS and fixed in 4% paraformaldehyde at 4℃ for 15 min. After removing the fixative, cells were washed three times (5 min per wash) with slight shaking and covered in cold methanol (-20℃) for 10 min to enhance permeability, and then were blocked in 1% BSA with 0.3% Triton X-100 (Sigma) for 2 h after washing. After dilution of antibodies with the blocking agent prepared above, cells were incubated overnight at 4℃ with NF-κB p65 mouse mAb (1:1,000; 6956; Cell Signaling, USA), followed by three washes (5 min each time) and incubation with anti-mouse IgG FITC (1:1,000; Abbkine, USA) for 1 h at RT. Cells were then washed and incubated for 10 min with 4,6-diamino-2-phenylindole (DAPI; Invitrogen, USA), and then washed with PBS two times. Cells were examined under a laser scanning confocal microscope (LSM710; Carl Zeiss, Germany).

Statistical Analysis

GraphPad Prism 5 (GraphPad Software Inc., CA, USA) was utilized to analyze data obtained by western blot, ELISA, and qRT-PCR. Statistical analyses were performed using one-way ANOVA tests. P-values <0.05 were considered to represent statistically significant differences.

 

Results

IL-10 Expressed on the Surface of Lactobacillus plantarum NC8

To predict surface probability, the three large transmembrane regions of psgA were evaluated. Because of the moderate size of the truncated protein, the mass of smaller regions between the second and the third large transmembrane regions appear to be the suitable sites for surface expression. Furthermore, except for surface probability, the antigenic index and hydrophilicity at the ~125th–135th, 180th–190th, and 210th–220th amino acids met our requirements for surface display. Based on our findings, the 1st-188th amino acid sequence of pgsA was selected as pgsA’ and gene fragments were obtained using customized primers (Table 1).

To induce the expression of IL-10 on the surface of L. plantarum NC8, IL-10-1 was fused to the 3’-end of pgsA or pgsA’ to yield pSIP409-pgsA (pgsA’)-IL-10 (Fig. 1B) before electroporation. Meanwhile, recombinant strains that harbored pSIP409-IL-10 or pSIP409-pgsA were also constructed. After western blot analysis, all target proteins could be observed on the membrane at the predicted sizes. However, the levels of the two bands with fusion proteins were significantly higher compared with the non-fusion one (Fig. 1E). Furthermore, the darkest band of 41 kDa that contained pgsA’-IL-10 represented the maximum expression of IL-10 (Fig. 1D, lane 3). A nonspecific band (~45 kDa) from L. plantarum-pgsA-IL-10 was also observed (Fig. 1D, lane 2), which may represent nonspecific immune reactivity between the sample and antibodies.

RAW264.7 Cells Treated with Recombinant L. plantarum Differ in Their Ability to Secrete Cytokines in Response to Poly(I:C) or LPS

Levels of Th1 cytokines in supernatants of RAW264.7 cells were detected by ELISA. As expected, Poly(I:C) or LPS stimulation of RAW264.7 cells induced the expression of pro-inflammatory cytokines. However, rIL-10 as a positive control could clearly inhibit the upregulation of Th1 cytokines in response to stimuli. In treated RAW264.7 cells, compared with higher IL-1β and IFN-γ production after the stimulation of Poly(I:C), production of TNF-α showed no apparent difference between the two stimuli. Nevertheless, expression levels of the other three cytokines were far lower than those of TNF-α after the two types of stimuli. Because of the increased production of Th1 cytokines by L. plantarum, the release of four factors was the most notable in cells co-cultured with L. plantarum-pgsA after the TLR ligand stimuli compared with the other treatments. Levels of these factors in RAW264.7 cells incubated with L. plantarum-10 were apparently lower than cells treated with L. plantarum-pgsA, but the indexes remained higher than for any other treatment. However, after co-culture with IL-10-expressing strains containing anchorins, different outcomes were obtained. After co-culture with L. plantarum-pgsA-IL-10 or L. plantarum-pgsA’-IL-10, levels of the four cytokines were significantly lower than for cells co-cultured with L. plantarum-IL-10 in most cases. In addition, cells co-cultured with strains harboring anchorins induced even less TNF-α (Fig. 2C and 3C) and IFN-γ (Fig. 2D) than the non-co-culture treatments (PBS). Interestingly, treatment with L. plantarum-pgsA’-IL-10 resulted in the lowest level of these four cytokines, which might be related to the highest level of IL-10 expression for strain, as measured by western blot assay.

Fig. 2.After co-culture with different types of recombinant L. plantarum and stimulation with Poly(I:C), the levels of four cytokines in the supernatant of RAW264.7 cells were measured by ELISA. *, **, and ***, significance of differences at p < 0.05, p < 0.01, and p < 0.001, respectively. Data represent the mean ± SEM of triplicate experiments.

Fig. 3.After co-culture with different types of recombinant L. plantarum and stimulation with LPS, the levels of four cytokines in supernatants of RAW264.7 cells were detected by ELISA. *, **, and ***, significance of differences at p < 0.05, p < 0.01, and p < 0.001, respectively. Data represent the mean ± SEM of triplicate experiments.

RAW264.7 Cells Treated with Recombinant L. plantarum Differ in Their Ability to Express IL-1β and IL-6 mRNAs in Response to Poly(I:C) or LPS

By qRT-PCR, most changes in gene expression were generally in accordance with ELISA data. In contrast with LPS treatments, expression levels of IL-1β and IL-6 mRNAs were relatively higher after stimulation with Poly(I:C), which indicates that RAW264.7 cells were more sensitive to Poly(I:C) than LPS. Furthermore, after the addition of Poly(I:C) or LPS, cells co-cultured with L. plantarum-pgsA’-IL-10 expressed the lowest levels of IL-1β and IL-6 mRNA transcripts among the groups stimulated with both TLR ligands, which was even lower than the groups stimulated with LPS alone (Fig. 4B). Despite the higher mRNA transcript levels of the two cytokines in the L. plantarum-pgsA-IL-10 groups, levels in the L. plantarum-pgsA-IL-10 and L. plantarum-pgsA’-IL-10 groups were dramatically lower than for cells cultured with L. plantarum-IL-10; these results were similar to those obtained by ELISA.

Fig. 4.After co-culture with different types of recombinant L. plantarum and stimulation with Poly(I:C) (A) or LPS (B), total RNA of RAW264.7 cells was prepared and converted to cDNA. The expression levels of IL-1β and IL-6 mRNA transcripts were detected by qRT-PCR; ** and ***, significance of differences at p < 0.01 and p < 0.001, respectively. Data are presented as the mean ± SEM of triplicate experiments.

NF-κB p65 Levels in RAW264.7 Cells Examined by Immunofluorescence Microscopy

NF-κB p65, also known as RelA, is a family of nuclear transcription factors that play critical roles in mediating the inflammatory responses of cells. Under a microscope, p65 expression levels were in direct proportion to the brightness (green). Using LSCM, we found that the cells generally released more p65 after treatment with Poly(I:C) than with LPS, which also indicated that these cells were more sensitive to stimulation with Poly(I:C). The p65 levels were not markedly increased after stimulation by both TLR ligands, and merged images of cell nuclei were more substantially dark blue except for the green spots around perinuclear compartments. Moreover, L. plantarum-pgsA groups had the maximum NF-κB p65 levels among all treatments, so the merged images were generally aquamarine blue. However, our data illustrated that IL-10 expressed by recombinant strains had certain inhibitory effects on the activation of NF-κB p65. In the merged images of L. plantarum-IL-10, compared with the dark green observed for the effects of Poly(I:C), cell nuclei were dark blue and had many green spots evoked by stimulation with LPS. As we observed no noticeable color differences among the non-co-cultured groups and L. plantarum-pgsA-IL-10 groups as well as L. plantarum-pgsA’-IL-10 groups, p65 levels of the latter two groups were similar to the groups of single stimulation with TLR ligands, especially the L. plantarum-pgsA’-IL-10 groups.

 

Discussion

Recently, Streptococcus pyogenes M6 protein [5], Lactobacillus brevis S-layer protein [1], Lactococcus lactis AcmA protein [26], and ice nucleation protein (INP) [10] have been used as anchoring motifs for lactic acid bacteria; however, pgsA is more stable and has a higher security in clinical use, so it has a wider range of applications for surface display on some types of Lactobacillus compared with other anchorins. Nevertheless, regarding the plasmids, the sequences of some anchored genes were so long that the length of downstream heterologous genes would be a limiting factor. To improve the efficiency of the construction of recombinant bacteria, especially those generated by electroporation, the anchorins are typically shortened. Li [20] attempted to remove the central repeat domains of INP, using the N-terminal and NC-terminal as an anchorin, and expression of the bioactive organophosphorus hydrolase on the surface of E. coli could occur effectively. Based on the same principle, we truncated pgsA by predicting the surface probability, antigenic index, and hydrophilicity of pgsA to generate pgsA’ and then constructed recombinant L. plantarum expressing IL-10. Importantly, the electroporation rate of L. plantarum-pgsA’-IL-10 was higher than that of L. plantarum-pgsA-IL-10 (data not shown). As verified by western blot assay, the recombinant strains with anchorin had higher IL-10 expression than the strains that lacked it. Surprisingly, L. plantarum-pgsA’-IL-10 had the highest expression level of IL-10, indicating that expression of exogenous proteins may be related to the full length of plasmids for these strains.

NF-κB includes five family members in mammals: p65 (RelA, NF-κB3), RelB, Rel (cRel), p105/p50 (NF-κB1), and p100/p52 (NF-κB2). These transcription factors play a crucial role in inflammatory and immune responses [9]. The family members p105 and p100 act as inhibitors of NF-κB (IκB), and after the process of phosphorylation, they are hydrolyzed to produce p50 and p52, respectively, by the ubiquitin-proteasome pathway [2]. In resting cells, p65 binds p50 (or p52) and IκB to form heterotrimers and then these NF-κB subunits are sequestered in the cytoplasm by IκB inhibitory proteins. Stimuli, such as pathogen-associated molecular pattern (PAMP) molecules, can induce the rapid disassociation of IκB by triggering IκB kinase (IKK) and the release of p65 to enter the nucleus where it regulates the expression of pro- and anti-inflammatory proteins [7,8]. Promoters of biomacromolecules (such as cytokines, chemokines, adhesion molecules, and colony stimulating factors) related to the inflammatory responses almost always contain binding sites for NF-κB, so the expression levels of the above proteins are regulated by the NF-κB signaling pathways. Certainly, Poly(I:C) or LPS, the ligands of TLR3 or TLR4, respectively, can also induce the activation and translocation of NF-κB in cells so that the expression of pro-inflammatory cytokines is increased [28].

However, because of the diverse properties of the strains, the regulatory processes of NF-κB p65 used by lactobacilli are dramatically diverse. Activation of NF-κB p65 and the expression of Th1 cytokines could be inhibited by some strains, such as L. casei 3260 [16]. In contrast to L. casei, L. plantarum can produce a large amount of Th1 cytokines and modulate the Th1/Th2 balance [24,29], which may be associated with TLR2 or TLR5 pathway activation by some surface components (e.g., peptidoglycan, lipoteichoic acid, and flagellin) of this strain [11]. This finding is consistent with our results that suggested that the strongest transcription and expression of Th1 cytokines and NF-κB p65 occurred in Poly(I:C)- or LPS-stimulated RAW264.7 cells pretreated with L. plantarum-pgsA.

Th2 cytokines, such as IL-10, can efficiently suppress pro-inflammatory cytokine secretion by monocytes-macrophages by blocking the phosphorylation of NF-κB and restricting the expression of related inflammatory mediator genes. In addition, IL-10 can also affect the expression of receptors associated with cytokines [30]. Moreover, monocytes can be deformed to osteoclasts with irregular shapes by receptor activator of nuclear factor κB ligand (RANKL) signaling through inducing the expression of NFATc1. However, IL-10 has a potent inhibitory effect on osteoclastogenesis at an early stage, which prevents the differentiation of osteoclast progenitors to preosteoclasts. Evans and Fox [6] confirmed that IL-10 could downregulate osteoclastogenesis by suppressing NFATc1 expression and nuclear translocation. Mohamed et al. [22] also found that IL-10 could maintain the basic form of monocytes by restraining the expression of c-Fos and c-Jun.

As representative Th1 cytokines secreted by RAW264.7 cells after stimulation with Poly(I:C) or LPS, IL-1β, IL-6, TNF-α, and IFN-γ can mirror the inflammatory responses of this types of monocyte-macrophages, which can be reduced by IL-10. In our present study, RAW264.7 cells appeared to be more sensitive to Poly(I:C) than LPS. The inhibitory effects of IL-10 were also validated by a positive control (rIL-10) and the IL-10-expressing strains (Figs. 2 and 3). Compared with cells pretreated with L. plantarum-IL-10, the transcription and expression levels of Th1 cytokines in the cells pretreated with the recombinant strains containing anchorins (especially L. plantarum-pgsA’-IL-10) were substantially lower after stimulation by Poly(I:C) or LPS. Levels of NF-κB p65 were also reduced by IL-10 expressed by L. plantarum-pgsA-IL-10 or L. plantarum-pgsA’-IL-10, even though there was no significant difference between them (Fig. 5). Based on our western blot findings, the reduction of Th1 cytokines might have a positive correlation with levels of surface-displayed IL-10 on these strains.

Fig. 5.Expression levels of NF-κB p65 in RAW264.7, measured by immunofluorescence microscopy. After co-culture with different types of recombinant L. plantarum and stimulation with Poly(I:C) (A) or LPS (B), cells were treated with NF-κB p65 mouse mAb(1:1,000) and anti-mouse IgG FITC(1:1,000) and the cell nucleus was stained with DAPI. Then, cells were examined using a laser scanning confocal microscope (magnification, ×400; scale bar, 200 μm).

In summary, our data demonstrated that both pgsA and pgsA’ could express heterologous proteins on the surface of L. plantarum, and the protein expression level and property of the recombinant strain with pgsA’ were greater than pgsA, which provides a useful tool for subsequent studies of heterogeneous protein expression on the surface of recombinant lactic acid bacteria. However, we only established the superior properties of the expression of heterologous proteins by recombinant L. plantarum containing pgsA’ in vitro, and the role played by recombinant bacteria in vivo may be more complex. Therefore, the different functions of heterologous proteins on strains displayed by pgsA and pgsA’ are still worth exploring in vivo. Furthermore, surface-displayed expression systems still have some problems that need to be solved. First, the growth rates of recombinant strains with anchorin (notably L. plantarum-pgsA’ expression systems) are significantly lower than the original strains. The production of novel recombinant strains cannot be improved by prolonging the incubation time because exogenous protein expression levels will gradually decrease after entering the stable growth period. Second, optimized foreign genes are more suitable for expression by a Lactobacillus expression system, but there are no effective methods to ensure the proper conformations of exogenous proteins. Third, plasmids for the expression of exogenous proteins usually depend upon antibiotic resistance genes (e.g., pSIP409 contains an erythromycin resistance gene). However, these genes can be integrated into the host genome, and the small amount of strains in feces may cause potential “gene pollution” in the environment. Despite the use of some “food-grade” resistance genes that have been applied as novel selective markers, our Lactobacillus expression system will still require extensive safety testing.

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