Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a remedial treatment modality for most hematologic malignancies. Its main complication, graft-
versus-host disease (GvHD) affects the skin, liver, and gastrointestinal tract. GvHD is mediated by donor T cells in the graft and leads to a high rate of transplantation-related mortality [
1,
2].
Tacrolimus (also known as FK506), a calcineurin inhibitor, is an anti-T-cell agent that is among the most widely used immunosuppressants [
3]. FK506 inhibits calcineurin phosphatase and calcium-dependent events by binding to FK506 binding protein 12 (FKBP12), thereby preventing the dephosphorylation of nuclear factor of activated T-cells (NFAT) family members responsible for transcription of the T-cell–activating cytokines interleukin-2 and -4 [
4‐
7]. However, the use of FK506 is associated with nephrotoxicity and can lead to dysbiosis [
8‐
10].
The altered intestinal microbiota of patients with GvHD is correlated with GvHD severity and pathogenesis [
11‐
13]. Intestinal bacteria of the order
Clostridiales are important mediators of intestinal homeostasis [
14‐
16] and the loss of intestinal bacteria of the genus
Blautia, a member of the order
Clostridiales, is associated with an increased mortality rate from GvHD [
17]. Therefore, controlling the intestinal microbiota composition may be useful for preventing and treating GvHD [
18].
Supplementation of probiotics may benefit gut-related immunity [
19]. In mice,
Lactobacillus rhamnosus reduced histologic inflammation and decreased the mortality rate post-transplantation [
20].
Lactobacillus species exert an anti-inflammatory effect in vitro, and elimination of
Lactobacillus species prior to allo-HSCT correlates with increased GvHD severity [
21,
22].
We hypothesized that treatment with the combination of L. acidophilus and FK506 would ameliorate inflammation and GvHD. We evaluated the effect of L. acidophilus plus FK506 on GvHD in vitro and in vivo.
Methods
Mice
C57BL/6 (B6, H-2 kb) and BALB/c (B/c, H-2kd) mice at 8–10 weeks of age were purchased from OrientBio (Sungnam, Korea). The Institutional Animal Care and Use Committee (IACUC) and Department of Laboratory Animal of the Catholic University of Korea (Songeui Campus) accredited the Korea Excellence Animal laboratory Facility from Korea Food and Drug Administration in 2017 and acquired full accreditation by AAALAC International in 2018. Animal procedures were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments of the IACUC of the School of Medicine of The Catholic University of Korea (Approval number: CUMC-2020-0177-01).
Patients
For this study, Liver transplantation (LT) patients were prospectively enrolled from a single LT clinic at Seoul St. Mary’s Hospital. The inclusion criteria were: more than 18 years old, liver transplanted more than three years ago; with 0–1 HLA-A, -B, and –DR mild mismatched; a high level of liver function with no history of rejection. Eight patients are being treated with CNIs including cyclosporine and tacrolimus. This study was approved by the Institutional Review Board of the Catholic University of Korea (KC19OESI0617).
Murine splenocyte and human peripheral blood mononuclear cell culture and stimulation in vitro
Spleens from B6 mice were homogenized and red blood cells were lysed with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM ethylenediaminetetraacetic acid; pH 7.2–7.4). Splenocytes were filtered through a cell strainer, centrifuged at 1300 rpm at 4 °C for 5 min, and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% [v/v] heat-inactivated fetal bovine serum (FBS). Human peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples. Human blood samples by adding phosphate-buffered saline (PBS) was placed onto a layer of 10 mL Ficoll Plaque Plus (GE Healthcare Life Sciences, Marlborough, MA, USA) and centrifuged at 2000 rpm at 20 °C for 30 min. After centrifugation, PBMCs were washed and maintained in RPMI 1640 medium containing 10% FBS. A single suspension was prepared, and 1 × 106 cells/well in 24-well flat bottom plates were cultured in the presence of plate-bound 0.5 μg/mL anti-CD3. Next, the cells were treated with L. acidophilus LA1 (100 ug/mL) and FK506 (0.3 nM for splenocytes; 1.0 nM for PB.
MCs) for 3 days. L. acidophilus was made inactive by heating at 80 °C, 30 min.
Murine T-cell isolation and conditioning of alloreactive T-cell responses
To purify mouse splenic CD4+-T cells and antigen-presenting cells (APCs), splenocytes were incubated with CD4-coated magnetic beads and isolated by magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). Splenic CD4+-T cells from B6 (or B/c) mice were used as stimulator cells in the context of allorecognition. APCs from B/c (or B6) mice were used as the responder cells. Aliquots of 2 × 105 CD4+-T cells (responders) were cultured with 2 × 105 irradiated (5000 cGy) stimulators in 96-well plates containing 200 μL/well of complete medium, followed by treatment with 100 ug/mL L. acidophilus and/or 0.3 nM FK506 (for splenocytes) at 37 °C in a humidified 5% (v/v) CO2/air atmosphere for 4 days. The cells were pulsed with 1 μCi of tritiated thymidine (3[H]-TdR) (NEN Life Science Products, Boston, MA, USA) 18 h before harvesting using an automated harvester (PHD Cell Harvester; Cambridge Technology, Cambridge, MA, USA), and enumerated using a β-counter (Packard TopCount NXT). Data are expressed as mean cpm values of triplicate samples ± SEM.
In vitro co-culture systems
To determine the blocking ability of alloreactive T-cell responses by L. acidophilus and/or FK506-induced Treg cells, we sorted the induced Treg cells and co-cultured them in alloreactive condition. Anti-CD3-stimulated CD4+-T cells were cultured with L. acidophilus, FK506 and combination of L. acidophilus and FK506. Three days later, cells of each condition were immunostained with anti-CD4 and anti-CD25 (eBioscience) and the induced Treg cells were sorted using a FACS Aria Fusion (BD Biosciences). Responder cells (2 × 105) and irradiated APCs (2 × 105) were co-cultured with each induced Treg cells (5 × 103; each induced Treg to responder cells ratio is 1:40) in 96-well plates for 4 days, pulsed with 1 μCi tritiated thymidine at 18 h before the end of the experiment, and counted using an automated harvester, and enumerated using a β-counter (Packard TopCount NXT). Data are expressed as mean cpm values of triplicate samples ± SEM.
Mouse bone marrow transplantation and scoring
To induce GvHD, splenocytes (5 × 10
6) and bone marrow cells (5 × 10
6) were isolated from donor (B6) mice and transplanted into recipient (B/c) mice by intravenous injection after 690 cGy irradiation. After induction of GvHD, recipient mice were orally administered
L.
acidophilus (1.6 × 10
10 CFU/kg) and/or FK506 (5 mg/kg) daily. Control GvHD mice received vehicle (saline) in the same manner. The experiments were performed in at least triplicate, with five mice per group. Survival after bone marrow transplantation (BMT) was monitored daily and GvHD severity was assessed twice weekly using a scoring system based on summing changes in weight loss, posture, activity, fur texture, and skin integrity. Clinical GvHD scores were assessed using a previously described scoring system [
23].
Flow cytometry
Murine splenic lymphocytes and PBMCs and human PBMCs were immunostained with fluorescently conjugated antibodies against CD4, CD8, CD25, IL-17, IFN-γ, Foxp3, and Fixable-dye (BD Biosciences, San Diego, CA, USA). Prior to intracellular staining, cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA) and treated with Golgistop (BD Biosciences). Intracellular staining was performed using a Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and Golgistop Kit (BD Biosciences). The transcription factor Foxp3 was stained using a Foxp3/Transcription Factor Staining Kit (eBioscience) following the manufacturer’s protocol. Flow cytometry was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed in FlowJo software (Tree Star).
Enzyme-linked immunosorbent assays
The concentrations of IFN-γ, IL-17, and IL-10 in culture supernatants of splenocytes and human PBMCs stimulated with anti-CD3 were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (Duoset; R&D Systems, Lille, France).
RNA sequencing
CD4+-T cells were isolated from B6 mice and subjected to T-cell activation conditions with or without L. acidophilus and FK506. Next-generation RNA-sequencing (RNA-seq) was conducted to quantify mRNAs and performed using a Novaseq 6000 (Illumina). Expression data were preprocessed using the range migration algorithm followed by quantile normalization. The RNA-seq data have been submitted in NCBI’s Gene Expression Omnibus (GEO) and is accessible through GEO Series Accession Number GSE174214.
Histological and immunohistochemical analyses
Mice were euthanized on day 42 after BMT and the organs were harvested, cryoembedded, and sectioned. Tissue specimens were fixed in 10% formalin buffer and embedded in paraffin. Sections (6 μm thick) were stained with hematoxylin and eosin and analyzed using a histologic scoring system. For immunohistochemical staining, sections were stained with primary antibodies against IFN-γ, IL-17, and IL-10 overnight at 4 °C, followed by a biotinylated secondary antibody and streptavidin-peroxidase mixture for 1 h (Thermo Fisher, San Diego, CA, USA). Color was developed by adding 3,3-diaminobenzidine (Dako, Carpinteria, CA, USA). A histopathology score was graded using the scoring system described by Kim et al. and Fukui et al. [
23,
24].
Real-time polymerase chain reaction
Total RNA was extracted using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized using the First-Strand cDNA Synthesis Kit (Dyne Bio). A LightCycler 2.0 instrument (software version 4.0; Roche Diagnostics) was used for PCR amplification with The SensiFAST™ SYBR Hi-ROX Kit (Bioline) following the manufacturer’s instructions. The following primers were used to amplify human genes: Foxp3, 5′-CAC TGC CCC TAG TCA TGG T-3′ (sense) and 5′-GGA GGA GTG CCT GTA AGT GG-3′ (antisense); and IL-10, 5′-CCA AGC CTT GTC TGA GAT GA-3′ (sense) and 5′-TGA GGG TCT TCA GGT TCT CC-3′ (antisense). Transcript levels were normalized to that of β-actin.
Statistical analysis
We performed one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test for multiple comparisons at a significance level of p < 0.05 using Prism ver. 5.01 software (GraphPad Software Inc., San Diego, CA, USA). Data are presented as means ± SEM.
Discussion
Tacrolimus (FK506) is an immunosuppressant used to prevent allograft and treat autoimmune diseases [
25‐
28]. However, immunosuppressive agents increase susceptibility to infections and alter the gut microbiota composition, the latter is associated with the severity and pathogenesis of GvHD [
10‐
13,
29]. For this reason, maintaining the optimal concentration of tacrolimus is important for patient survival; however, determining the optimum concentration is hampered by interindividual variation in tacrolimus efficacy [
30‐
32]. We hypothesized that
L.
acidophilus could ameliorate the side effects of FK506, and thus mitigate GvHD following MHC-mismatched bone marrow transplantation.
Probiotics supplementation is widely used in clinical practice, and promotes re-growth of beneficial species, ameliorating diseases such as IBD, colitis, RA, and osteoporosis [
33‐
37]. IFN-γ-producing T cell such as Th1 and Tc1, IL-17-producing T cell such as Th17 and Tc17 are crucial mediators of GvHD [
38‐
43] and GvHD is associated with a reversed proportion of Tregs [
44,
45].
Lactobacillus species, probiotics widely used as health supplements, are commensal bacteria that reduce inflammation by stimulating lymphocytes via IL-10 [
46,
47]. Furthermore,
L.
acidophilus, specifically the LA1 strain, is known to interact with intestinal cells to activate pathways that repair defective intestinal barriers and effectively treat intestinal inflammation [
48]. In this study, the combination treatment significantly decreased the population of Th1, Tc1, Th17 and Tc17 cells (Figs.
1a, c,
5a, c,
6a and b) as well as the IFN-γ and IL-17 concentrations, but significantly increased the population of Treg cells and the IL-10 concentration, compared with FK506 alone (Figs.
1a, b,
5b, c and
6b) in murine and human. Moreover, the alloreactive T-cell response was decreased significantly by the combination treatment (Fig.
1d). However, the transcript levels of Foxp3 and IL-10 in human PBMCs were significantly increased by the combination treatment (Figs.
5d,
6b). Further study is required to confirm that combination treatment of
L. acidophilus with FK506 can modulate Tc1 and Tc17 cell in vivo and human in vitro.
Dysbiosis of the gut microbiota influences immune tolerance and triggers autoimmune and inflammatory diseases by causing an imbalance of T cell subsets [
49,
50].
L.
acidophilus has immunomodulatory properties such as modulating Th17 and Treg cell populations.
L.
acidophilus induced production of Treg cells and IL-10, but suppressed that of IL-17, in splenocytes [
35]. In addition, the immunoregulatory function of Tregs is used in the therapeutic strategies for autoimmune diseases such as SLE, RA and GvHD because mechanisms of Treg cells that have been modulation of cell proliferation [
51‐
54]. In this study, combination treated-induced Treg suppressed the T cell proliferation (Fig.
1e) and the combination treatment reduced the effector T-cell population by modulating Th17, mTOR, and JAK-STAT signaling, and upregulating Treg-related genes, compared to
L.
acidophilus and FK506 alone (Fig.
2). In particular, Treg-related genes,
Nt5e,
Foxp3,
Ikzf2,
Nrp1 and
Itgb8, are up-regulated by combination treatment of
L. acidophilus and FK506.
Nt5e, also known as CD73, is known to have immunosuppressive potential through restricting inflammatory immune responses [
55,
56].
Foxp3 is essential for the maintenance of Treg and
Nrp1 is required to maintain Treg stability and function [
57‐
59]. The deficiency of
Ikzf2 (as known as Helios) and
Itgb8 cannot control the expansion of pathogenic T cells during active inflammation [
60,
61]. It could be suggested that the combination showed a greater effect than single treatment of FK506 because of the Treg-related genes. Therefore,
L.
acidophilus improved the immunological imbalance caused by FK506 by decreasing and increasing the Th17 and Treg cell populations in vitro.
L.
acidophilus reduced the serum IL-6 and IL-17 levels and downregulated IL-17A expression, but upregulated CD25, Foxp3, and TGF-β expression in a murine model of allergy [
62]. Also,
L.
acidophilus leads to the Treg-Th17 cell balance by inhibiting Th17 cells and promoting Treg cells in ovariectomized mice [
63]. We used
L.
acidophilus because of its immunoregulatory activity, as confirmed in this study by its in vivo and in vitro immunoregulatory effects on Th17 and Treg cells in a GvHD model. Indeed, the combination treatment improved the clinical scores of mice with GvHD. Moreover, the combination therapy reduces the Th1 and Th17 cell populations compared with vehicle after 4 weeks in vivo (Fig.
3b). In GvHD target tissues, such as skin and liver, the levels of proinflammatory cytokines were significantly decreased and that of an anti-inflammatory cytokine was significantly increased by the combination treatment (Fig.
4). Therefore, the combination treatment suppressed inflammation and pathogenic cell activity.
Taken together, the results show that treatment with L. acidophilus or FK506 exerted similar effects, which were of lesser magnitude than those of the combination treatment. In other words, L. acidophilus served as a supplement to low-dose FK506 in a GvHD model and showed synergistic effects in this study.
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