Oral Administration of Mice with Cell Extracts of Recombinant Lactococcus lactis IL1403 Expressing Mouse Receptor Activator of NF-kB Ligand (RANKL)

Wild-type L. lactis IL1403 was used as the host strain for recombinant protein production, and grown in M17 medium (MBcell, Seoul, Korea) supplemented with 5 g/L of glucose (M17G). Recombinant L. lactis IL1403 was grown in M17G media with chloramphenicol (5 μg/mL) and erythromycin (5 μg/mL) at 30°C.

Mouse RANKL sequence was used in Knoop and Kim’s studies (Kim et al., 2015; Knoop et al., 2009). To secrete the target protein, the signal peptide of USP45 (van Asseldonk et al., 1993) was added to the N-terminus, and to detect the expressed protein, a his-tag (his6x) was added to the C-terminus of the target gene. The designed amino acid sequence was codon-optimized using DNAWorks v3.2.4 (Hoover and Lubkowski, 2002) based on the L. lactis IL1403 codon usage table. The insert fragment was synthesized by Macrogen (Seoul, Korea). The insert fragment was shown in Supplementary Fig. S1. The plasmid vector pILPtuf.Mb was used as the backbone (Kim et al., 2009). Insert and vector were ligated at the NdeI and XhoI restriction sites and transformed into wild-type L. lactis IL1403 (WT_CE) competent cells. Vector construction is shown in Fig. 1A.

Fig. 1. Schematic diagram for construction of recombinant mRANKL expression vector system and detection of target gene expression. (A) Plasmid vector system. (B) Western blot for detecting mRANKL from cell extracts (intracellular) and cell-free culture supernatants (extracellular), lane 1: Cell extracts of Lactococcus lactis IL1403; lane 2: Cell-free culture supernatant of L. lactis IL1403; lane 3: Cell extracts of recombinant L. lactis IL1403; lane 4: Cell-free culture supernatant of recombinant L. lactis IL1403. mRANKL, mouse receptor activator of NF-kB ligand.

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Wild-type and recombinant L. lactis strains were cultured in 50 mL of M17G broth without and with antibiotics, respectively. The optical density of each sample was measured at a wavelength of 600 nm every 1 h for 12 h and 24 h after inoculation.

The expression of mRANKL from recombinant L. lactis was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Wild type and recombinant L. lactis were cultured in M17G broth without and with antibiotics at 30°C for 10 h, respectively. For preparation of cell extracts, 10 mL of cultured cells were harvested by centrifugation at 12,300×g for 1 min and lysed using a bead beater with 0.5 g sterilized glass beads (0.5 mm) and 200 μL 1×phosphate buffered saline (PBS). The secreted proteins were collected from 10 mL of cultured cell-free supernatant (0.2 μm filtered) through trichloroacetic acid precipitation, and dissolved in 200 μL 1×PBS for SDS-PAGE and western blot analysis. To quantify the amount of mRANKL produced, 18 kDa commercial recombinant His-tagged human calmodulin (Merck KGaA, Darmstadt, Germany) was used for construction of the standard curve using amounts of 1.5, 1, and 0.5 μg.

RAW 264.7 cells were seeded on cell culture dishes in Dulbecco’s modified Eagle’s medium (DMEM; Capricorn, Düsseldorf, Germany) with 10% fetal bovine serum (FBS; Capricorn) and 1% penicillin and streptomycin (P/S) at a density of 2×10⁶ cells/mL. After seeding for 4 h, the medium was changed to DMEM with 10% FBS, 1% P/S, and 30 ng/mL macrophage colony-stimulating factor with crude cell extracts of wild-type or recombinant L. lactis, that were prepared as described above. The group treated with 1×PBS as control was named PBS, and groups treated with cell extracts from wild type L. lactis IL1403 and mRANKL producing L. lactis were named WT_CE and mRANKL_CE, respectively. According several dose test, finally, the cell extracts containing 90 ng/mL of mRANKL used in this study, and the same amount of cell extracts of WT_CE were used. Commercial mouse RANKL (Abcam, Hanam, Korea) was added to the medium at a concentration of 60 ng/mL.

After 72 h of treatment, the medium was replaced with fresh media, and the cells were incubated for another 72 h. Total RNA was extracted using TRIzol^® (ThermoFisher, Seoul, Korea) according to the manufacturer’s instructions, and cDNA was synthesized using PrimeScript^TM RT reagent Kit (Takara Bio, Shiga, Japan). qRT-PCR was conducted using TB Green^® Premix Ex Taq^TM (Tli RNaseH Plus, Takara Bio) with specific primers TRAP-F:5′-GCGACCATTGTTAGCCACATACGG-3′; TRAP-R:5′-CGCCCAGGGAGTCCTCAGATCCAT-3′ and primers for the housekeeping gene GAPDH-F:5′-AACTTT GGCATTGTGGAAGGGCTC-3′; GAPDH-R:5′-AAGGCCATGCCAGTGAGCTTC-3′. mRNA levels were normalized to those of GAPDH (Kim et al., 2015).

Four-week-old BALB/c mice were randomly assigned to the three groups (PBS, WT_CE, and mRANKL_CE). Before oral administration, 300 μL of neutralizing reagent (1.5% NaH₂CO₃) was administered to prevent gastric acidity. After 30 min, 100 μL 1×PBS was fed to the PBS group, and the cell extracts from 2.5×10⁸ CFU wild type L. lactis IL1403 and cell extracts containing mRANKL (4.2 μg) from 2.5×10⁸ CFU of recombinant L. lactis were fed to WT_CE and mRANKL_CE groups, respectively. All groups were fed for seven consecutive days and sampled on the eighth day (Supplementary Fig. S2).

Peyer’s patches of small intestine samples were extracted to determine the abundance of mature M cells by measuring the GP2 mRNA expression level. Isolation of RNA and synthesis of cDNA was same as above method. qRT-PCR was conducted using TB Green^® Premix Ex Taq^TM (Tli RNaseH Plus, Takara Bio) with M cell-specific primers GP2-F:5′- GATACTGCACAGACCCCTCCA-3′; GP2-R:5′- GCAGTTCCGGTCATTGAGGTA-3′ (Kusunose et al., 2020), and primers for the housekeeping gene GAPDH-F:5′-AACTTTGGCATTGTGGAAGGGCTC-3′; GAPDH-R:5′-AAGGCCATGCCAGTG AGCTTC-3′. mRNA levels were normalized to those of GAPDH (Kim et al., 2015).

Nine ileum samples of the same size (1 cm) were extracted from the same position (distal ileum). Total RNA was isolated from the tissues using the Maxwell (Promega) method. One milligram of total RNA was processed to prepare the mRNA sequencing library using the MGIEasy RNA Directional Library Prep Kit (MGI), and sequencing was performed using the MGIseq system.

After a quality check, raw reads were trimmed to 100 bp prior to mapping to the mouse reference genome GRCm38/mm10 using HiSat2 (version 2.1.0; Pertea et al., 2016). Following read alignment, counts assigned to features were computed using the featureCounts (version 2.0.3; Liao et al., 2014). Differentially expressed genes (DEGs) were identified using the DESeq2 (v1.24.0; Love et al., 2014). A gene was considered to be a DEG with a fold change>1.45 and adjusted p-value<0.1 using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995). Gene Ontology (GO) enrichment analysis was performed using in-house Perl scripts. The significantly enriched GO terms were determined by Fisher’s exact test with p<0.05 and odds ratio>1.

Genomic DNA was extracted from fecal samples using a NucleoSpin Soil kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. DNA samples (5 ng) were used to amplify the 16S ribosomal RNA V4 region using Takara Ex-taq DNA polymerase (Takara Bio) with universal primer sets (Forward:5′-GGACTACHVGGGTWTCTAAT-3′ and R:5′-GTGCCAGCMGCCGCGGTAA-3′; Han et al., 2018). After amplification, all the samples were normalized to 50 ng per sample. A DNA library was then constructed and sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA), generating 2×300 bp paired-end reads.

To analyze the gut microbiome, de-multiplexed and pre-processed sequence reads were imported into Quantitative Insights Into Microbial Ecology (QIIME2, version 2021.2; Bolyen et al., 2019). Barcode and primer removal, quality control, amplicon sequence data correction, and de-replication, were performed using the DADA2 (Callahan et al., 2016). Feature tables and representative sequence files were merged for downstream analysis using QIIME2. Taxonomic classification was performed using the SILVA 132 database with 99% identity, based on the V4 16S region. All classification was performed within QIIME2 and was assigned using the naïve Bayesian algorithm available in the sklearn python library. For phylogenetic diversity analysis, alpha and beta diversities were calculated using the q2-diversity plugin, and included Faith’s phylogenetic diversity and weighted and unweighted UniFrac distances. Differential abundance analysis of microbiota was performed using an in-house Perl script. We considered a p-value<0.05 to indicate statistical significance.

Serum samples were collected on the eighth day, and concentration was measured using a Calcium Colorimetric Assay Kit (BioVision, Milpitas, CA, USA).

Statistical analysis was performed using an in-house Perl script and R (version 4.1.0) language. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for determining statistical significance.

To confirm the expression of mRANKL, intracellular and secreted proteins were precipitated and analyzed by western blotting. No mRANKL was detected in the wild-type L. lactis samples. In recombinant L. lactis, intracellular and secreted mRANKL proteins were detected with expected size of 23.86 and 20.88 kDa, respectively (Fig. 1B). According to the standard curve, recombinant L. lactis produced 2.1 μg/mL of mRANKL in intracellular fraction (Supplementary Fig. S3).

To examine the physiological characteristics of wild type and the recombinant L. lactis, patterns of growth were measured. As shown in Supplementary Fig. S4, recombinant L. lactis showed a slightly delayed growth rate compared to the wild type; after 10 h though, these two strains showed similar growth patterns.

To validate the biological activity of mRANKL, RAW 264.7 cells were used to observe the stimulation of RANK-RANKL signaling. As shown in Supplementary Fig. S5, treatment with culture media containing commercial RANKL and mRANKL_CE significantly (p<0.05) increased TRAP mRNA expression levels compared with those in the PBS and WT_CE groups.

Cell extracts containing recombinant mRANKL were orally administered to mice for seven consecutive days, and the expression of mature M cells marker in Peyer’s patches was analyzed by qRT-PCR. As shown in Supplementary Fig. S6, the GP2 mRNA expression levels in the mRANKL_CE group were significantly (p<0.05) higher than those in the PBS and WT_CE groups. In addition, body weight gain was measured between day 1 and day 8 of the experiment, and the results showed that oral administration of WT_CE and mRANKL_CE had no significant effect on body weight (Supplementary Table S1). Our results showed that recombinant mRANKL from recombinant L. lactis is biologically active and does not cause significant changes in body weight.

To compare the gene expression between the PBS, WT_CE, and mRANKL_CE groups, RNA-Seq analysis of the mouse small intestine was performed. The WT_CE and mRANKL_CE groups were compared with the PBS group, with a cut-off fold change>1.45 and adjusted p-value<0.1. No DEGs were identified between the PBS and WT_CE groups. Between the PBS and mRANKL_CE groups, 63 DEGs, including 53 upregulated and 10 downregulated DEGs, were identified (Fig. 2 and Supplementary Table S2). Between the WT_CE and mRANKL_CE groups, 192 DEGs were identified, including 169 upregulated and 23 downregulated DEGs (Supplementary Table S2). These results indicate that cell extracts from WT_CE had no effect on mouse small intestinal gene expression, and only mRANKL had an effect.

Fig. 2. Venn diagram of co-up and co-downregulated genes. (A) Upregulated genes that control (PBS) versus treatment (WT_CE and mRANKL_CE). (B) Downregulated genes that control (PBS) versus treatment (WT_CE and mRANKL_CE). mRANKL_CE, recombinant Lactococcus lactis expressing mouse receptor activator of NF-kB ligand; WT_CE, wild-type L. lactis IL1403; PBS, phosphate buffered saline.

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To analyze the gene enrichment of DEGs, GO analysis were performed with a threshold of p<0.05 and odds ratio>1. Between the PBS and mRANKL_CE groups, 49 upregulated and 9 downregulated DEGs were annotated with GO terms. The upregulated GO terms included ‘de novo’ protein folding (p<0.001), response to unfolded protein (p<0.001), regulation of bone resorption (p<0.01), heat shock protein binding (p<0.001), and calcium ion binding (p<0.05; Table 1 and Supplementary Table S3). Between the WT_CE and mRANKL_CE groups, 159 upregulated and 22 downregulated DEGs were annotated with GO terms. The upregulated GO terms included calcium ion binding (p<0.001), calmodulin binding (p<0.001), and ‘de novo’ protein folding (p<0.05; Table 2 and Supplementary Table S3).

To compare the gut microbial diversity, the alpha and beta diversities of the three groups from normalized microbiome sequencing reads were investigated. For alpha diversity, three indices including observed features, Shannon and Faith’s phylogenetic diversity (Faith PD) were measured. None of the three indices showed any significant differences among the three groups (Supplementary Fig. S7). For beta diversity, principal coordinate analysis (PCoA) of unweighted and weighted UniFrac distances was performed. There were no significant differences among the three groups (Supplementary Fig. S8).

To compare the differences in major gut microbial taxa among the three groups, the microbial composition in these three groups were examined. The overall microbial composition in the gut was not significantly different among the three groups. However, the genera Lactobacillus (p<0.05), Sphingomonas (p<0.01) and Acinetobacter (p<0.01) differed significantly (Fig. 3 and Supplementary Table S4). Moreover, there was no significant difference among the three groups in the genus Lactococcus (Fig. 3 and Supplementary Table S4). These results showed that feeding cell extracts with or without mRANKL did not have a significant effect on the gut microbiome.

Fig. 3. The relative abundance (%) of genus among that PBS, WT_CE and mRANKL_CE three groups. (A) Lactococcus. (B) Lactobacillus. (C) Sphingomonas. (D) Bacteroides. For significance tests, a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test were used. PBS, phosphate buffered saline; WT_CE, wild-type Lactococcus lactis IL1403; mRANKL_CE, recombinant L. lactis expressing mouse receptor activator of NF-kB ligand.

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After RANKL administration, serum calcium levels were measured to compare the changes in calcium concentration among the three groups. There was no significant difference in serum calcium levels among the three groups (Supplementary Fig. S5).