Mesenchymal stem cell-derived exosome-educated macrophages alleviate systemic lupus erythematosus by promoting efferocytosis and recruitment of IL-17+ regulatory T cell

Female C57BL/6 mice and MRL/lpr mice at age 6–8 weeks were purchased from The Jackson Labs (Bar Harbor, USA). Mice were placed in the SPF facility of the animal center of Nanjing University School of Life Sciences and reared at room temperature 25 ± 2 ℃, relative humidity 65 ± 2%, and a 12 h light / dark cycle. The mice were reared in an animal facility for one week before the experiment.

Mouse nephritis model was induced in an 8-week-old female C57BL/6 mouse by a single intraperitoneal injection of 500 μl of pristane oil (2,6,10,14-tetramethylpentadecane, Sigma-Aldrich, USA).

Kidneys were processed as previously described [20], and the cell pellet was resuspended in D-hanks containing 0.5% BSA and 2 mM EDTA. Anti-F4/80 coated MACS beads (Miltenyi, Germany) were used to sort macrophages.

Macrophages were resuspended and blocked for 30 min in D-hanks with 2% BSA. The cells were then incubated with 1:500 diluted FITC-anti-F4/80, or FITC-anti-CD206 and PE-anti-CD86, or FITC-anti-B7H4 or FITC-anti-CD138 antibody (BioLegend; San Diego, CA) for 45 min (room temperature and protected from light). The cells were then washed with D-hanks twice and resuspended in D-hanks with 2% BSA for flow cytometry analysis. The cells incubated with FITC-Isotype or PE-Isotype (BioLegend) were used as the isotype control. The gating strategy is shown in Additional file: sFig 1A. 1

Cells were washed with PBS twice and lysed. Protein samples were extracted and then separated on a 4–12% precast Bis–Tris gel (Thermo Fisher Scientific). Later proteins were transferred to polyvinylidene difluoride (PVDF, Rancho Palos Verdes, USA) membranes. The membrane was blocked for 1 h in TBST (with 5% nonfat milk) and incubated with primary antibodies overnight at 4 ℃. Proteins were evaluated by using primary anti-iNOS and anti-Arg-1 (Cell Signaling, MA) antibodies. Membranes were washed with TBST 3 times (a total of 45 min). The washed membranes were incubated with second antibodies for 1 h, at room temperature. Enhanced chemiluminescence reagents were used to illuminate protein and ImageQuant ™ LAS 4000 Luminescent Image Analyzer (GE Healthcare; Chicago, IL) was used to expose protein bands.

The conditioned medium (CdM) was collected to evaluate the concentration of cytokines. Levels of certain cytokines, including IFN-γ, IL-1β IL-6, IL-12, GM-CSF, IL-10, TGF-β, and M-CSF, were evaluated by using ELISA kits (R&D Systems). The procedures for the cytokine and growth factor assays were carried out according to the manufacturer's instructions. The Molecular Devices SpectraMax spectrophotometer (Marshall Scientific, Hampton, NH) was employed to measure the concentration of cytokines.

ROS was detected by using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Grand Island, NY) according to the manufacturer’s instructions. Briefly, the macrophage-conditioned medium was collected, and the absorbance was checked by using the Molecular Devices SpectraMax spectrophotometer. The results were normalized to a standard curve.

Mouse kidney epithelial cells (TCMK-1) were incubated with heat shock (45 ℃, 10-min) and cultured for 4 h to induce apoptosis as described previously. The percentage of apoptotic cells was evaluated by flow cytometry. The apoptotic cells were washed with PBS twice and labeled with pHrodo Red (Life Technologies). Then, the 1 × 10 [6] cells were added to 1 × 10⁶ macrophages to determine the phagocytotic capacity. After, being incubated for 2.5 h at 37 ℃, the macrophages were washed three times with ice-cold D-hanks and stained with FITC rat anti-mouse CD11b antibody (BioLegend, USA) for 45 min at 4 ℃. Then, the cells were harvested and examined by flow cytometry.

The cell suspension was blocked for 30 min in D-hanks with 2% BSA. The cells were then incubated with 1:500 diluted FITC-anti-Foxp3, or PE-anti-CD3, or PE/APC-anti-CD4, or APC-anti-IL-17 antibody (BioLegend; San Diego, CA) for 45 min (room temperature and protected from light). The cells were then washed with D-hanks twice and resuspended in D-hanks with 2% BSA for flow cytometry analysis. The cells incubated with FITC-Isotype or PE-Isotype or APC-Isotype (BioLegend) were used as the isotype control. The gating strategy is shown in Additional file: sFig 1B. 1

The Mouse Bone Marrow-derived Mesenchymal Stem Cells were purchased from Cyagen (CA, USA). The cells were cultivated in an OriCell Mouse MSC Growth Medium (Cyagen) containing 100 units/ml of penicillin and 100 μg/ml of streptomycin at 37 ℃, 5% CO₂. The medium was changed to fresh medium every 2–3 days.

Exosomes were isolated from the BM-MSC medium by qEV exosomes isolation kit (iZON Science; MA, USA) according to the manufacturer’s protocol. Briefly, the conditioned medium was collected and centrifuged at 2,500 × g for 20 min to remove unattached cells and debris. A 100 kDa molecular weight cutoff in a centrifugal filter (Millipore Sigma; MA, USA) was employed to concentrate the medium. The concentrated medium was transferred to a size-exclusion chromatography qEV unit. The liquid that contained exosomes was collected and then reconcentrated by using a 10 kDa molecular weight cutoff centrifugal filter (Millipore Sigma).

The isolated exosomes were lysed, and the concentration of total proteins was measured by using a BCA Protein Assay Kit (Thermo Fisher Scientific, NJ, USA) according to the manufacturer’s instructions.

An LM10 NTA device (Malvern; Amesbury, UK) was used according to the manufacturer’s instructions to analyze the characteristics of microparticles that were isolated.

Exosomes were labeled by using an ExoGlow-Protein EV Labeling Kit (Green, System Biosciences; Palo Alto, CA) according to the manufacturer’s instructions. Briefly, the exosomes were incubated with the green fluorescent dye at 37 ℃ for 20 min and then incubated at 4 ℃ overnight. The next day, the labeled exosomes were isolated and resuspended in the macrophage growth medium. Macrophages were incubated in the medium that contained 50 µg/ml exosomes for various times (3 h, 6 h, 12 h, or 24 h). The macrophages were harvested and incubated with a 1:10,000 diluted Hoechst 33,342 (Thermo Fisher Scientific) to label the nucleus. The fluorescent pictures were taken under fluorescent microscopy (Olympus DP70, Japan). For flow cytometry analysis, the cells were collected and analyzed by using a Guava easyCyte flow cytometer (Millipore Sigma). The results were further analyzed by using FlowJo software.

The glomerular mesangial expansion was scored as follows: 0, no any expansion; 1, 1–10% glomeruli with mild expansion; 2, 11–25% glomeruli with moderate expansion; 3, 26–50% glomeruli with severe expansion; 4, 50% glomeruli being sclerotic. Twenty glomeruli were examined and scored for each mouse. A pathologist who was blinded to the sample identities performed the evaluation and scoring.

Sections of 5-mm thickness of mouse frozen kidney tissues were blocked with 10% FBS and incubated with primary antibodies labeled with FITC, rabbit polyclonal anti-IgG, IgA, IgM, C3, and C1q (DAKO, USA). The images were captured under the Leica microscope (DM5000B).

For IgG, IgA, IgM, C3, and C1q a pathologist blinded to the sample identities examined the glomeruli and scored staining intensity by 0 (no staining), 1 (weak staining), 2 (positive staining), and 3 (strongly positive).

The conditioned medium (CdM) of macrophage was collected to determine the concentration of CCL20 using ELISA kits (R&D Systems). The procedures were carried out according to the manufacturer's instructions. The Molecular Devices SpectraMax spectrophotometer (Marshall Scientific, Hampton, NH) was employed to measure the concentration of CCL20.

Mouse naïve T cell was isolated from the spleen by using Mouse Naïve CD4⁺ T Cell Isolation Kit (R&D Systems) according to the manufacturer's instructions. Briefly, the mouse spleen was teased apart to generate a single cell suspension in D-Hanks’ supplemented with 10% bovine serum. The cells were washed and resuspended in cold 1X MagCellect Buffer at 1 × 10⁸ concentration. Then, the cells were incubated with Mouse Naïve CD4⁺ T Cell Biotinylated Antibody Cocktail,

Total RNA was extracted from cells or exosomes using TRIzol Reagent (ThermoFisher) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using TaqMan miRNA probes (Applied Biosystems, USA) according to the manufacturer’s instructions. Briefly, total RNA was reverse transcribed to cDNA using AMV reverse transcriptase (ThermoFisher) and a stem-loop RT primer (Applied Biosystems) or RT primer. Real-time PCR was performed using a TaqMan PCR kit and an Applied Biosystems 7900 Sequence Detection System (Applied Biosystems). All reactions, including no-template controls, were run in triplicate. After the reaction, the CT values were determined using fixed threshold settings. miRNA expression in cells or tissues is normalized to U6 snRNA or Cel-miR-39.

The procedures practiced here followed the National Institutes of Health guidelines for recombinant DNA research. All recombinant lentiviruses used in this study were purchased from the AMSBIO (Abingdon, UK). For transduction, macrophages were incubated with the Lenti/PREmiR-16 (PREmiR-16) and Lenti/PREmiR-21 (PREmiR-21) or Lenti/PREmiR-Cont (PREmiR-Cont) at a multiplicity of infection (MOI) of 5. BMMSCs were incubated with Lent/ZIPmiR-16 (ZIPmiR-16) and Lent/ZIPmiR-21 (ZIPmiR-21) to deplete miR-16 and miR-21 levels in the cells. Lent/ZIPmiR-Cont (ZIPmiR-Cont) was used as control.

All values are shown as the means ± SDs. All experiments were repeated at least 4 times (n = 4) unless otherwise noted. Student’s t test analysis was performed to analyze all data. Only p < 0.05 was considered significant.

There is a known role of macrophages in the development of SLE [21]. To further study the phenotype of macrophages in SLE mice, macrophages from the kidney of 18-week-old MRL/lpr mice were collected. Our results demonstrated that infiltration of F4/80⁺ macrophages in SLE mice was higher than in the wild-type mice (control) (Fig. 1A). Flow cytometry analysis revealed that macrophages collected from SLE mice presented higher levels of CD86, a pro-inflammatory polarization marker, but presented low levels of CD206, an anti-inflammatory polarization marker, when compared to macrophages from control mice (Fig. 1B). Both groups of macrophages failed to express B7H4 and CD138 (Fig. 1C). Immunoblot results indicated increased protein levels of iNOS while the level of Arg-1 decreased in the macrophages from SLE mice (Fig. 1D). These results suggest that macrophages in the kidney of MRL/lpr mice are polarized to a specific phenotype defined as CD86^hiCD206⁻B7H4⁻CD138^loiNOS^hiArg-1⁻. Cytokine analysis of the conditioned medium of macrophages collected from SLE mice exhibited upregulation of pro-inflammatory cytokines IFN-γ, IL-1β, IL-6, IL-12, and GM-CSF. In contrast, anti-inflammatory cytokines IL-10, TGF-β, and M-CSF were downregulated in these macrophages (Fig. 1E). Also, reactive oxygen species (ROS) are active contributors to pro-inflammatory conditions; we found that ROS levels were upregulated in the macrophages from SLE mice (Fig. 1F). Many studies support that an accumulation of apoptotic cells plays a crucial role in SLE progression. Efferocytosis activity analysis demonstrated that macrophages from SLE mice exhibit a lower efferocytosis activity than control mice (Fig. 1G). T_reg cells are potent immune suppressors and were observed to be downregulated in SLE patients. Our results demonstrated that Foxp3⁺ T cells (T_reg) decreased in the SLE mouse kidney (Fig. 1H). However, the upregulation of T helper (Th) 17 cells that facilitate inflammation and SLE development was detected in the SLE mouse's kidney (Additional file 1: sFig 2A). Further, the accumulation of pro-inflammatory macrophages and Th17 cells resulted in the downregulation of anti-inflammatory cytokines but the upregulation of pro-inflammatory cytokines in the serum from SLE mice (Additional file 1: sFig 2B and 2C). These data suggest that macrophages from MRL/lpr mice are polarized to a pro-inflammatory phenotype and cause a reduced efferocytosis activity and failed apoptotic cell clearance that further induces the steady deterioration of SLE.

Exosomes are microparticles secreted from cells. In this study, we utilized a nanoparticle tracking analysis to determine the characteristics of the exosomes that were isolated from BMMSCs. The isolated particles (~ 40 nm) fell within the normal size distribution of exosome (Additional file 1: sFig 3A). Immunoblot analysis for known exosome markers, TSG-101 and Alix, was identified and detected (Fig. 2A). Green fluorescent-labeled exosomes were used to investigate uptake efficiency. Fluorescent images (Additional file 1: sFig 3B) and flow cytometry analysis (Additional file 1: sFig 3C) revealed that the internalization of exosomes into macrophages increased to 90% after 24 h. To study the role of exosomes from BMMSCs in alleviating SLE under pathophysiological conditions, 18-week-old MRL/lpr mice were tail vein injected exosomes from BMMSCs on day 0, day 3, and day 7; PBS was used as control. On day 10, the kidney, lung, liver, heart, and spleen were harvested from all mice. Our results showed that tail vein injected exosomes were significantly accumulated in the liver and kidney (Fig. 2B and Additional file 1: sFig 4). Results of H&E and PAS staining demonstrated that the exosome treatment downregulated immune cell infiltration in the renal interstitium (Fig. 2C) and reduced glomerular mesangial expansion (Fig. 2D); PBS was used as a control. Electron microscope observation and immunofluorescence further confirmed that exosome treatment lessened dense deposits (Fig. 2E) and immune deposits including IgG, IgM, C3, and C1q (Fig. 2F and 2G) in the glomerular mesangial and endocapillary, respectively. Interestingly, although there was no difference in the infiltration of macrophages in the kidney of mice that received exosomes or PBS (Fig. 2H and Additional file 1: sFig 5), T cell infiltration was significantly downregulated in the kidney of the exosome-treated mouse (Fig. 2I and Additional file 1: sFig 6). Moreover, exosome treatment promoted kidney-infiltrated macrophage M2-like polarization, which was defined by a high level of CD206 (Fig. 2J and Additional file 1: sFig 7). Altogether, our results demonstrated that exosomes from BMMSCs alleviated SLE nephritis in lupus mouse by increasing kidney accumulation of M2-like macrophage and reducing the kidney infiltration of T cells.

Exosomes, especially from stem cells, are reported to mediate many biofunctions including macrophage polarization [17]. To study the role of exosome from BMMSCs in macrophage polarization, macrophages were collected from 18-week-old MRL/lpr mice kidneys and incubated with 50 µg/ml exosomes derived from BMMSC for 3 days. Flow cytometry analysis demonstrated that CD86 on the macrophages was downregulated, while CD206 was upregulated (Fig. 3A). Moreover, compared to PBS-treated macrophages, exosome-treated macrophages expressed more B7H4 and CD138 (Fig. 3B). Additionally, exosome treatment downregulated iNOS but upregulated Arg-1 in the macrophages (Fig. 3C). Furthermore, the conditioned medium from macrophages treated with the exosomes was analyzed for their secretory content. ELISA results detected elevated levels of CCL20, a chemokine normally found ligated to CCR6 on T cells (Fig. 3D). Meanwhile, pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-6, IL-12, GM-CSF were decreased, and anti-inflammatory cytokines including IL-10, TGF-β, and M-CSF were increased (Fig. 3E). We found that there was a significant reduction in reactive oxygen species (ROS) levels in exosome-treated macrophages, this further confirmed that macrophages had shifted to an anti-inflammatory phenotype (Fig. 3F). Again, an efferocytosis assay was used to determine the efferocytosis activity of macrophages. Exosome-treated macrophages efferocyted increasingly more apoptotic cells when compared to PBS-treated macrophages (Fig. 3G). More interestingly, conditioned medium collected from exosome-treated macrophages induced more Naïve T cells to differentiate into T_reg cells (Fig. 3 H). These results suggest that pro-inflammatory macrophages from SLE mice can be polarized to a defined anti-inflammatory phenotype, CD206⁺, Arg-1⁺, B7H4⁺, and CD138⁺ if pre-treated with exosomes. These macrophages secrete high levels of anti-inflammatory cytokines, efferocyte more apoptotic cells, and even evoke naïve T cells' transition to T_reg cells.

To understand the underlying molecular mechanisms of exosome-mediated macrophage polarization, total RNA from exosome-treated macrophages was isolated and subjected to miRNA profiling. Total RNA from PBS-treated macrophages was used as a control. The results of microarray analysis indicated that 20 miRNAs from exosome-treated macrophages were 2 times more upregulated when compared to PBS-treated macrophages (p < 0.05, Additional file 1: sFig 8). Among these upregulated miRNAs, 6 miRNAs were reported to promote macrophage anti-inflammatory polarization. The 6 miRNAs were validated using qRT-PCR analysis (Fig. 4A). In the context of this study, we naturally hypothesized that miRNAs aid in anti-inflammatory polarization of exosome-treated macrophage and these miRNAs may be delivered by exosomes. Interestingly, qRT-PCR results showed that miR-16 and miR-21 were enriched in the exosomes from BMMSCs (Fig. 4B).

It has been reported that miR-16 promotes macrophage anti-inflammatory polarization by targeting PDCD4 and miR-21 similarly does the same by targeting PTEN. To investigate the role of miR-16 and miR-21 in the SLE macrophage anti-inflammatory polarization, macrophages were transduced using a lentivirus-based miRNA overexpression vector to upregulate cellular miR-16 and miR-21. Macrophages transduced with lentivirus control were used as a control. After transduction, the levels of miR-16 and miR-21 were elevated significantly (Additional file 1: sFig 9A). Interestingly, immunoblot analysis demonstrated that the levels of iNOS, PDCD4, and PTEN decreased, but the level of Arg-1 increased in the macrophages that overexpressed miR-16 and miR-21 (Fig. 4C and Additional file 1: sFigure 9B). Further, miR-16 and miR-21 overexpression induced an increase in CD206, B7H4, and CD138 surface markers on the macrophages (Fig. 4 D). More important, there was a significant downregulation of pro-inflammatory cytokines (Fig. 4 E) and ROS (Additional file 1: sFig 9C), but upregulation of anti-inflammatory cytokines (Fig. 4 F) and chemokine CCL20 (Fig. 4 G) was observed in the miRNAs overexpressed macrophages. Results from the efferocytosis assay concluded that by overexpressing miR-16 and miR-21 in macrophages, it incurred increased efferocytosis of apoptotic cells than untransduced macrophages (Fig. 4H). Similarly, miRNA-overexpressed macrophages induced more production of T_reg cells (Fig. 4I).

Next, we depleted miR-16 and miR-21 in exosomes. To do this, we isolated exosomes from BMMSCs that were transduced with a lentivirus-based anti-miRNAs vector; the level of miR-16 and miR-21 was downregulated in the exosomes (Fig. 5A). When macrophages were treated with the miRNAs-knockdown exosomes, the exosomes treatment induced upregulation of miR-16 and miR-21 in macrophage was abolished (Additional file 1: sFig 10A). Furthermore, exosomes induced downregulation of iNOS, PDCD4, and PTEN, as well as upregulation of Arg-1 in the macrophages was also attenuated when the miRNAs were depleted (Fig. 5B and Additional file 1: sFigure 10B). Flow cytometry analysis also demonstrated that the expression of CD206, B7H4, and CD138 upregulated by exosomes was eliminated (Fig. 5C). The concentration of pro-inflammatory cytokines in macrophages that received miRNAs-knockdown exosomes was much higher than macrophages that received normal exosomes. Conversely, the concentration of anti-inflammatory cytokines was found to be much lower in miRNAs-knockdown exosomes treated macrophages (Fig. 5D). Knockdown miR-16 and miR-21 also abolished exosome-mediated CCL20 upregulation (Fig. 5 E) and increased ROS level (Additional file 1: sFig 10C). As expected, when compared to normal exosome treatment, the miRNAs-knockdown exosome-treated macrophages efferocyted less apoptotic cells (Fig. 5F) and induced fewer T_reg cells (Fig. 5G). Additionally, depleting miR-425-5p (Additional file 1: sFigure 11A), which also enriched in the exosome, did not erode the effect of the exosome. The exosome-treated macrophages were polarized to a phenotype similar to the unmodified exosome-treated one (Additional file 1: sFigure 11B). Our findings provide evidence that exosomal miR-16 and miR-21 play a critical role in exosome-induced SLE macrophage anti-inflammatory polarization by regulating the PDCD4 and PTEN pathways.

To understand the underlying mechanisms of exosomes from BMMSCs in alleviating SLE under pathophysiological conditions, C57BL/J mice were subjected to intraperitoneal injections with pristane to induce mouse lupus nephritis. Four months post-injection was defined as day 0. PBS, 100 µg exosomes from BMMSCs that were transduced with lenti-anti-miR-Cont (Exo/Anti-Control) or exosomes from BMMSCs that were transduced with lenti-anti-miR-16 and lenti-anti-miR-21 (Exo/Anti-miRNAs) were tail vein injected into the mouse on day 0, day 3, and day 7. On day 10, the kidneys, spleen, and blood were harvested from all mice. Our results showed that exosome treatment reduced pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-6, IL-12, IL-17a, and GM-CSF expression (Fig. 6A) but elevated anti-inflammatory cytokines such as IL-10, TGF-β, and M-CSF expression (Fig. 6B) in mouse serum. Knockdown of miR-16 and miR-21 in the exosomes obliterated the anti-inflammatory effect of exosomes. Interestingly, there were no significant differences in kidney macrophage infiltration among the three groups (Fig. 6C). However, the phenotype of infiltrated macrophages was drastically different. The expression of CD206 (Additional file 1: sFig 12A), Arg-1 (Additional file 1: sFig 12B), B7H4, and CD138 (Fig. 6D) were promoted, while the expression of CD86 and iNOS were decreased in the macrophages that collected from Exo/Anti-Control treated mouse kidney. The secretions of macrophages were also shifted, pro-inflammatory cytokines (Additional file 1: sFig 13A) and ROS (Additional file 1: sFig 13B) were downregulated but anti-inflammatory cytokines (Additional file 1: sFig 13C) and chemokine CCL20 (Fig. 6E) were upregulated in mice injected with the Exo/Anti-Control (Control). Efferocytosis assay further confirmed that macrophages collected from Exo/Anti-Control treated mouse kidneys ingested more apoptotic cells (Fig. 6F). Again, depleting miR-16 and miR-21 in the exosome attenuated the effects of exosomes. It has been well established that T cells dominate the development of SLE. Our results revealed that the infiltration of CD3⁺CD4⁺ pan T cells was decreased in the kidney of mice that received Exo/Anti-Control injection (Fig. 6G). Surprisingly, the percentages of IL-17⁺ T cells in pan T cells were similar among all three groups (Fig. 6H). Considering that IL-17⁺ T cells can be divided into two groups: (1) Th 17 cells which are verified to accelerate the progression of SLE, and (2) IL-17⁺ T_reg cells which are IL-17⁺ and Foxp3⁺ and thought to contribute to the revitalizing of SLE. Our data demonstrated that in the Exo/Anti-Control treated mouse kidney, most (> 70%) IL-17⁺ T cells were Foxp3⁺ and IL-17⁺ T_reg cells. Moreover, our data showed the IL-17⁺ T_reg cells infiltrated in the kidney were CCR6⁺ (Additional file 1: sFig 14). In support of our previous data, we demonstrated that macrophages can be polarized by exosomes and secrete a large amount of CCL20, a ligand of CCR6. It is reasonable to postulate these IL-17⁺ T_reg cells were recruited or differentiated by exosome polarized macrophages. Moreover, Exo/Anti-Control treatment also increased IL-17⁺ T_reg cells in the mouse spleen, compared to Exo/Anti-miRNAs (Additional file 1: sFig 15). The CCR6⁺ IL-17⁺T_reg cells enrichment was significantly impaired when using miRNAs depleted exosome. Our in vivo data revealed that exosome injection reduced the inflammatory response in an induced mouse lupus nephritis model by polarizing macrophages to an anti-inflammatory phenotype. These specific polarized macrophages alleviated pristane-induced mouse lupus nephritis by secreting anti-inflammatory cytokines, engulfing apoptotic cells, and recruiting/inducing more IL-17⁺ T_reg cells.

All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Atlanta University Center and Nanjing University and complied with the NIH guidelines for the care and use of laboratory animals.

Not applicable.

The authors indicate no potential conflicts of interest.

The data that support the findings of this study are available from the corresponding author upon reasonable request.